TUPLER Rossella

Almost all FSHD patients carry rearrangements occurring in a 3.3 kb tandemly repeated sequence (D4Z4) located at the 4q subtelomeric region. D4Z4 reduced alleles have been associated to FSHD. Each repeated unit sequence contains heterochromatic DNA. By performing a set of experiments aimed at studying the role of D4Z4 in regulating gene transcription we characterized the protein binding activity of D4Z4, and the protein binding element within D4Z4 (D4Z4 Binding Element). Through biochemical purification methods and mass spectrometry microsequencing, we isolated three proteins, YY1, HMGB2B, and nucleolin that we believed to be part of a multi-protein complex binding D4Z4. Through several experiments we demonstrated that the three proteins were specifically binding D4Z4 in vitro and in vivo. Reduction of YY1, HMGB2, and nucleolin protein level resulted in over-expression of the 4q35 gene FRG2. This seminal work laid the basis for all the subsequent studies on FSHD molecular mechanism and on the molecular organization of the 4q35 region.

FSHD is characterized by great clinical variability and incomplete penetrance sometimes with unexpected mode of inheritance. To create tools for the systematic clinical study of FSHD families we created a evaluation form to collect clinical information and designed a standard protocol that quantifies muscle weakness by combining the functional evaluation of six muscle groups affected in FSHD and discovered that the current diagnostic molecular marker for FSHD diagnosis is a common polymorphism. We also established that gender and degree of kinship contribute to FSHD development in relatives carrying the FSHD molecular signature. Our results suggest that the genetic basis of FSHD, which is remarkably heterogeneous, should be revisited since this has important implications for genetic counseling and prenatal diagnosis of at-risk families. This large cohort of FSHD families provides a massive source of information that will support the validation of the generality of our findings and facilitate the translation of our study to general practice.

Guidelines for FSHD diagnosis are based on the molecular features of the D4Z4 locus at 4q35, even though great clinical variability can be associated with these molecular features, including healthy carriers from the general population. We established a novel methodology to approach myopathic patients with suspected FSHD and collected clinical and molecular data systematically in a large database that constitutes the basis of the Italian National Registry for FSHD. Presently we have promoted an international initiative to constitute a Global Registry for FSHD and generate an international consensus on FSHD diagnosis that will lay the basis for trial readiness for FSHD

Three genes, FRG2, FRG1, ANT1, were found overexpressed at the 4q35 region in biopsies from FSHD patients. We generated transgenic mice independently overexpressing these genes. Among them, mice overexpressing FRG1 display an overt dystrophic phenotype with features of disease. FRG1 is a RNA binding protein and its overexpression causes the aberrant splicing of numerous genes. We demonstrated that the aberrant splicing of Troponin T determines reduced response to Ca++ and strength of mouse muscle fibers and it is predominant in muscle from FSHD patients. At present FRG1 transgenics are the only mouse model mimicking the FSHD muscle pathology. They have been used to test possible therapeutic interventions in FSHD and to study genetic interactions.

When I first started studying FSHD in 1992, I analyzed chromosomal structural anomalies involving the 4q35 region to refine the FSHD locus. I employed molecular cytogenetics and basic molecular biology techniques and demonstrated that haploinsufficiency of the FSHD locus does not cause FSHD. I also established that FSHD can be expressed differently in presence of identical genetic background. In addition I conducted a pioneer study of differential gene expression in muscles of subjects affected by FSHD and controls. All these studies have laid the ground for the studies that have been conducted in the FSHD field.