Importance: Facioscapulohumeral muscular dystrophy (FSHD) is considered an autosomal dominant disorder, associated with the deletion of tandemly arrayed D4Z4 repetitive elements. The extensive use of molecular analysis of the D4Z4 locus for FSHD diagnosis has revealed wide clinical variability, suggesting that subgroups of patients exist among carriers of the D4Z4 reduced allele (DRA). Objective: To investigate the clinical expression of FSHD in the genetic subgroup of carriers of a DRA with 7 to 8 repeat units (RUs). Design, Setting, and Participants: This multicenter cross-sectional study included 422 carriers of DRA with 7 to 8 RUs (187 unrelated probands and 235 relatives) from a consecutive sample of 280 probands and 306 relatives from the Italian National Registry for FSHD collected between 2008 and 2016. Participants were evaluated by the Italian Clinical Network for FSHD, and all clinical and molecular data were collected in the Italian National Registry for FSHD database. Data analysis was conducted from January 2017 to June 2018. Main Outcomes and Measures: The phenotypic classification of probands and relatives was obtained by applying the Comprehensive Clinical Evaluation Form which classifies patients in the 4 following categories: (1) participants presenting facial and scapular girdle muscle weakness typical of FSHD (category A, subcategories A1-A3), (2) participants with muscle weakness limited to scapular girdle or facial muscles (category B, subcategories B1 and B2), (3) asymptomatic or healthy participants (category C, subcategories C1 and C2), and (4) participants with myopathic phenotypes presenting clinical features not consistent with FSHD canonical phenotype (category D, subcategories D1 and D2). Results: A total of 187 probands (mean [SD] age at last neurological examination, 53.5 [15.2] years; 103 [55.1%] men) and 235 relatives (mean [SD] age at last neurologic examination, 45.1 [17.0] years; 104 [44.7%] men) with a DRA with 7 to 8 RUs and a molecular diagnosis of FSHD were evaluated. Of 187 probands, 99 (52.9%; 95% CI, 45.7%-60.1%) displayed the classic FSHD phenotype, whereas 86 (47.1%; 95% CI, 39.8%-54.3%) presented incomplete or atypical phenotypes. Of 235 carrier relatives from 106 unrelated families, 124 (52.8%; 95% CI, 46.4%-59.7%) had no motor impairment, whereas a small number (38 [16.2%; 95% CI, 9.8%-23.1%]) displayed the classic FSHD phenotype, and 73 (31.0%; 95% CI, 24.7%-38.0%) presented with incomplete or atypical phenotypes. In 37 of 106 families (34.9%; 95% CI, 25.9%-44.8%), the proband was the only participant presenting with a myopathic phenotype, while only 20 families (18.9%; 95% CI, 11.9%-27.6%) had a member with autosomal dominant FSHD. Conclusions and Relevance: This study found large phenotypic variability associated with individuals carrying a DRA with 7 to 8 RUs, in contrast to the indication that a positive molecular test is the only determining aspect for FSHD diagnosis. These findings suggest that carriers of a DRA with 7 to 8 RUs constitute a genetic subgroup different from classic FSHD. Based on these results, it is recommended that clinicians use the Comprehensive Clinical Evaluation Form for clinical classification and, whenever possible, study the extended family to provide the most adequate clinical management and genetic counseling.

Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary myopathy causally linked to reduced numbers (≤8) of 3.3 kilobase D4Z4 tandem repeats at 4q35. However, because individuals carrying D4Z4-reduced alleles and no FSHD and patients with FSHD and no short allele have been observed, additional markers have been proposed to support an FSHD molecular diagnosis. In particular a reduction in the number of D4Z4 elements combined with the 4A(159/161/168)PAS haplotype (which provides the possibility of expressing DUX4) is currently used as the genetic signature uniquely associated with FSHD. Here, we analyzed these DNA elements in more than 800 Italian and Brazilian samples of normal individuals unrelated to any FSHD patients. We find that 3% of healthy subjects carry alleles with a reduced number (4-8) of D4Z4 repeats on chromosome 4q and that one-third of these alleles, 1.3%, occur in combination with the 4A161PAS haplotype. We also systematically characterized the 4q35 haplotype in 253 unrelated FSHD patients. We find that only 127 of them (50.1%) carry alleles with 1-8 D4Z4 repeats associated with 4A161PAS, whereas the remaining FSHD probands carry different haplotypes or alleles with a greater number of D4Z4 repeats. The present study shows that the current genetic signature of FSHD is a common polymorphism and that only half of FSHD probands carry this molecular signature. Our results suggest that the genetic basis of FSHD, which is remarkably heterogeneous, should be revisited, because this has important implications for genetic counseling and prenatal diagnosis of at-risk families.