An historical account
Facioscapulohumeral muscular dystrophy (FSHD) is a hereditary human myopathy, affecting groupes of muscles in the face and shoulder, mainly caused by the contraction of a macrosatellite array on chromosome 4q35. Despite recent advances on the molecular cascade initiated by this genetic abnormality, the mechanism by which it triggers the disease represents one of the most enigmatic conundrums for human geneticists and remains incompletely understood. Work with genetically engineered mutant mouse models, and on a collaboration with clinicians and human geneticists allowed us proposing the involvement of a novel genetic mechanism to pathophysiology of this disease.
As we were working on mechanisms of neuromuscular development, we discovered by chance that one of the genes we were working on, was potentially a good candidate to explain features of FSHD.
During development, muscle formation involves an event of collective migration of the muscle precursors. During this step of cell migration, coupling of the polarity of each individual cell to that of its neighbors is necessary for the cohesion of the collective trajectory, and for the precision of muscle shapes. Such coupling requires function of the FAT1 protocadherin, an adhesion molecule known for its implication in various morphogenetic processes. Constitutive ablation of the murine Fat1 gene leads to alterations of the shape of subsets of muscles in the face and shoulder region. These abnormalities of muscle structure and shape are followed by an early onset regionalized muscle wasting in Fat1-deficient mice surviving to adult stages. Selective ablation of Fat1 in muscle precursors before their migration reproduces in part those shape phenotypes, indicating that Fat1 is necessary in the migrating muscle cells.
The map of muscles affected by loss of Fat1 functions in mouse prefigures with an astonishing precision the topography of muscles affected in FSHD, a hereditary human myopathy, which most frequent form, FSHD1, is caused by a genetic abnormality carried by chromosome 4. In spite of spectacular recent advances in understanding of the molecular cascade initiated by this genetic defect, with identification of the transcription factor DUX4 as the main pathogenic agent in FSHD, how this genetic mistake leads to the very specific panoply of clinical symptoms still remained obscure. Strikingly, in human, the FAT1 gene lies near the locus affected in FSHD. Earlier studies investigating the potential implication of such proximal genes has disregarded FAT1 as a candidate. However, in addition to the similarity of muscle symptoms, mice lacking Fat1 surviving to adulthood also display non muscular phenotypes reminiscent of other symptoms of FSHD, such as vascular abnormalities in the retina. These elements led us to consider the provocative possibility that a partial alteration of FAT1 gene functions might have a link with this human myopathy.
Through a series of studies conducted in human, we have observed lowering of FAT1 expression levels (RNA and protein) in muscle but not brain of fetal cases diagnosed with the classical form of FSHD, FSHD1. Furthermore, we have found that DUX4, the transcription factor overexpressed in FSHD1 and FSHD2, is capable of repressing FAT1 expression in cultured human muscle cells. Finally, we have identified in the human FAT1 locus, deletions of a putative regulatory element of the FAT1 gene, predictive of a depletion of its expression in some specific tissue types, these deletions being overrepresented among FSHD patients as compared to a population of healthy subjects. Thus, while complete inactivation of Fat1 gene function in mice is lethal at birth and involves defects in tissues not affected in FSHD (thus being more severe than FSHD), selective ablation of Fat1 in tissues involved in FSHD, such as muscles, represents a unique means to mimic a crucial set of FSHD symptoms in mice, and to reinforce our knowledge on the pathogenesis of this complex disease
Figure : Loss of function of the Fat1 gene alters the shape of selective subsets of muscles in the face and shoulder, also affected in FSHD. (Left) Map of human muscles subject to degeneration in FSHD patients. Affected muscles are represented in dark purple. Top: face vue; Bottom: back vie. (Right) Skeletal muscles are visualized in mid gestation mouse embryos (14.5 days of development) owing to the blue staining, corresponding to expression of a specific marker detected through a color reaction. (Top) wild type embryo; (Bottom) embryo deficient for the functions of the Fat1 gene. The differences between these two embryos are represented by yellow dotted lines indicating reduced subcutaneous muscles, and with red dotted lines, indicating supernumerary muscles found in mutants. These differences, detected around the eye, in subcutaneous muscles of the face, in the region between proximal forelimb and the scapular area, reproduce precisely the map of muscles affected in FSHD. Observation of these abnormal muscles at higher magnification shows the appearance of missoriented muscle fibres in mutant muscles. © Francoise HELMBACHER
2015: New support for the link between FAT1 and FSHD
The possibility of a link between FAT1 alterations and FSHD was investigated in collaboration with Pr. N. Levy and M. Bartoli, La Timone, Marseille. Having found reduced FAT1 expression levels in fetal FSHD muscles and identified deletions of a putative regulatory enhancer, predicted to cause tissue-specific depletion of FAT1, which co-segregate with FSHD (Caruso 2013), we next wondered whether we could obtain direct evidence in humans, that mutations in the FAT1 exome could be uniquely associated with FSHD symptoms, even in absence of classical FSHD1 or FSHD2 genetic abnormalities. For this, we (Marc Bartoli, Francesca Puppo) studied patients which were diagnosed as FSHD based on they symptoms, but for which genetic testing excluded FSHD1 and 2. We call these patients FSHD-like. This study has identified a series of heterozygous point mutations, either perturbing splicing of FAT1, or leading to deleterious amino-acid changes, found in FSHD-like patients carrying neither pathogenic 4q35 alterations nor mutations in the other FSHD modifier gene SMCHD1 (Puppo et al, Human Mutation 2015). Thus, FAT1 is a compelling novel FSHD modifier gene, which partial loss-of-function (tissue-specific or /function-specific) is sufficient to recapitulate FSHD-like symptoms, even in absence of other FSHD-causing genetic abnormalities, and which deregulation was found to co-occur with FSHD.
Puppo et al, Human Mutation, 2015. Figure 1A&B. Functional validation using minigene splicing assay, that some of the FAT1 variants identified in FSHD-like patients interfere with RNA splicing, causing either complete exon skipping (A), or incomplete integration of a given exon. Such splicing errors are expected to lead to a truncated FAT1 protein, missing a portion of the extracellular domain, thus interfering with its functions.
Caruso N., Herberth B., Bartoli M., Puppo F., Dumonceaux J., Zimmermann A., Denadai S., Lebossé M., Roche S., Geng L., Magdinier F., Attarian S., Bernard R., Maina F., Levy N. and Helmbacher F. (2013). Deregulation of the protocadherin gene FAT1 alters muscle shapes: implications for the pathogenesis of Facioscapulohumeral dystrophy. PLoS Genet. (2013) Jun;9(6):e1003550. | PMID: 23785297 | doi: 10.1371/journal.pgen.1003550 |
Puppo F., Dionnet E., Gaillard MC., Gaildrat P., Castro C., Vovan C., Bertaux K., Bernard R., Attarian S., Goto K., Nishino I., Hayashi Y., Magdinier F., Krahn, M., Helmbacher, F., Bartoli, M, and Levy, N. (2015). Identification of variants in the 4q35 gene FAT1 in patients with a Facioscapulohumeral dystrophy (FSHD)-like phenotype. Human Mutation, 2015, 23 JAN. DOI: 10.1002/humu.22760 | PMID 25615407 |
Mariot V, Roche S, Hourdé C, Portilho D, Sacconi S, Puppo F, Rameau P, Caruso N, Delezoide AL, Desnuelle C, Bessières B, Collardeau S, Feasson L, Maisonobe T, Magdinier F, Helmbacher F., Mouly V, Butler-Browne G. Dumonceaux J. Correlation between low FAT1 expression and early affected muscle in FSHD. Annals of Neurology, 2015, MAY 28.