ARTICLE Large-Scale Population Analysis Challenges the Current Criteria for the Molecular Diagnosis of Fascioscapulohumeral Muscular Dystrophy Isabella Scionti, 1 Francesca Greco, 1 Giulia Ricci, 2 Monica Govi, 1 Patricia Arashiro, 3 Liliana Vercelli, 4 Angela Berardinelli, 5 Corrado Angelini, 6 Giovanni Antonini, 7 Michelangelo Cao, 6 Antonio Di Muzio, 8 Maurizio Moggio, 9 Lucia Morandi, 10 Enzo Ricci, 11 Carmelo Rodolico, 12 Lucia Ruggiero, 13 Lucio Santoro, 13 Gabriele Siciliano, 2 Giuliano Tomelleri, 14 Carlo Pietro Trevisan, 15 Giuliana Galluzzi, 16 Woodring Wright, 17 Mayana Zatz, 18 and Rossella Tupler 1,19, * 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 partic- ular 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 combi- nation 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. Introduction Facioscapulohumeral muscular dystrophy (FSHD [MIM 158900]), a common myopathy, has a prevalence of 1 in 20,000. 1,2 The disease is characterized by weakness of selec- tive muscle groups and wide variability of clinical expres- sion. 1,3,4 The onset of the disease is in the second or third decade of life and usually involves the weakening of facial and limb-girdle muscles. The mode of inheritance of classical FSHD is considered to be autosomal dominant, with complete penetrance by age 20. 4,5 No biochemical, histological, or instrumental markers are available to inde- pendently confirm a specific FSHD diagnosis that remains mainly clinical. The FSHD genetic defect does not reside in any protein- coding gene. 6 Instead, FSHD has been genetically linked to the reduction of an integral number of tandem 3.3-kb D4Z4 repeats located on chromosome 4q35. 7,8 Although nearly identical D4Z4 sequences reside on chromosome 10q26, 9 only subjects with a reduced number of D4Z4 repeats on chromosome 4, but not chromosome 10, develop FSHD. 10–12 Based on these results, p13E-11 EcoRI alleles larger than 50 kb (R11 D4Z4 repeats) originating from chromosome 4 have been considered normal, whereas alleles of 35 kb or less (%8 D4Z4 repeats) have been considered diagnostic for the disease. 8,13 Because there are individuals with reduced D4Z4 alleles that do not have clinical signs of FSHD, 14,15 it has been pro- posed that additional DNA sequences flanking the D4Z4 repeat array are necessary for disease development. 16–18 These studies concluded that D4Z4 reduction is pathogenic only in a few genetic backgrounds, which include a specific simple sequence length polymorphism (SSLP) proximal to the D4Z4 repeat and the 4qA polymorphism distal to 1 Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena 41125, Italy; 2 Department of Neuroscience, Neurological Clinic, University of Pisa, Pisa 56126, Italy; 3 Program in Genomics, Division of Genetics, Informatics Program, Children’s Hospital, The Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA; 4 Department of Neuroscience, Center for Neuromuscular Diseases, University of Turin, Turin 10126, Italy; 5 Unit of Child Neurology and Psychiatry, IRCCS ‘‘C. Modino’’ Foundation, University of Pavia, Pavia 27100, Italy; 6 Department of Neurosci- ences, University of Padua, Padua 35129, Italy; 7 Department of Neuroscience, Salute Mentale e Organi di Senso, S. Andrea Hospital, University of Rome ‘‘Sapienza,’’ Rome 00189, Italy; 8 Center for Neuromuscular Disease, University ‘‘G. d’Annunzio,’’ Chieti 66013, Italy; 9 Neuromuscular Unit, IRCCS Foundation Ca ` Granda Ospedale Maggiore Policlinico, Dino Ferrari Center, University of Milan, Milan 20122, Italy; 10 Unit of Muscular Pathology and Immunology, Neurological Institute Foundation ‘‘Carlo Besta,’’ Milano 20133, Italy; 11 Department of Neurosciences, Universita ` Cattolica Policlinico A. Gemelli, Rome 00168, Italy; 12 Department of Neurosciences, Psychiatry and Anaesthesiology, University of Messina, Messina 98125, Italy; 13 Department of Neurological Sciences, University ‘‘Federico II,’’ Naples 80131, Italy; 14 Department of Neurological Sciences and Vision, University of Verona, Verona 37134, Italy; 15 Department of Neurological and Psychiatric Sciences, University of Padua, Padua 35100, Italy; 16 Molecular Genetics Laboratory of UILDM, Lazio Section, IRCCS Santa Lucia Foundation, Rome 00179, Italy; 17 Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; 18 Human Genome Research Center, Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of Sa ˜o Paulo, Sa ˜o Paulo 05508-090, Brazil; 19 Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA *Correspondence: rossella.tupler@umassmed.edu DOI 10.1016/j.ajhg.2012.02.019. Ó2012 by The American Society of Human Genetics. All rights reserved. 628 The American Journal of Human Genetics 90, 628–635, April 6, 2012