Facioscapulohumeral muscular dystrophy (FSHD): Advanced diagnostics and extended molecular insights

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited dystrophic
myopathy characterized by progressive and irreversible weakness, mostly
beginning in the facial, shoulder and upper arm muscles. There is no cure for
FSHD and treatment options are limited. FSHD is caused by contraction of the
D4Z4 repeat (FSHD1), or by mutations in SMCHD1 or DNMT3B (FSHD2), both
resulting in D4Z4 chromatin relaxation and leading to the misexpression of the
transcription factor DUX4 in muscle. Due to the complex genetic mechanism, FSHD
genetic testing is cumbersome and currently not amenable to current routine
(for example, next generation sequencing) technologies. Recent studies show
that it is possible to isolate epithelial and stem cells from midstream urine
and that these cells, directly, or after transdifferentiation into myogenic
cells, can be used for the diagnosis of a number of limb girdle muscular
dystrophies. FSHD has not yet been tested, but DUX4 detection in cells from
urine could provide an appealing alternative for FSHD diagnostics. In addition,
our FSHD mouse models reveal relatively high DUX4 levels in the epidermal layer
of the skin (epithelial cells; likely keratinocytes), and preliminary studies
suggest that this expression pattern reflects that of FSHD patients. To
simplify diagnosis and because of the limited invasiveness, we therefore
propose to assess whether epithelial or stem cells from urine, buccal brush
biopsies or skin punch biopsies can be used as an alternative for FSHD
diagnosis. In addition to serving a diagnostic purpose, identification of novel
stem or somatic cell populations expressing DUX4 (without apparent detrimental
effect as seen in muscle) will provide a novel source of cells that can help us
to understand the basic pathological mechanism of FSHD. Furthermore, detection
of DUX4 (and detection of the target genes DUX4 regulates) in any of these
tissues may also serve as a biomarker for disease severity or in upcoming
clinical trials with drugs that aim to suppress DUX4. Next to DUX4, cytokines
may serve as biomarkers for FSHD development and progression, as an increasing
number of studies point towards a role for the immune system in FSHD
pathogenesis. Therefore, we also propose to measure cytokine levels in blood
samples from FSHD patients, healthy individuals, and patients with an unrelated
neuromuscular disease with the goal to identify novel biomarkers that can be
used for natural history studies and future clinical trials.

To determine whether DUX4 can be detected in epithelial or stem cells isolated
from midstream urine and/or buccal brush biopsies and/or skin punch biopsies of
FSHD patients. Hereby we will assess whether any of these biomaterials can be
used as an attractive alternative for FSHD diagnosis. The secondary objectives
are to investigate the role and regulation of DUX4 in these cell populations in
order to get a better understanding of the pathological mechanisms underlying
FSHD, and to identify novel biomarkers for FSHD development and severity.

Exploratory, case-control observational study

As stated in the introduction, FSHD is an irreversible and disabling disease
which leads to functional impairment. Genetic testing is cumbersome and
diagnosis of FSHD subtypes (FSHD1, FSHD2) requires different testing methods.
DUX4 detection in either urine, skin punch biopsies, or buccal brush biopsies
offer attractive new diagnosis methods. The generation of cellular models, like
iPS cells, will lead to more insight in the disease mechanism and facilitate
pre-clinical therapeutic tests. Finally, the identification of cytokines that
may serve as biomarkers for development and progression of the disease is
important for natural history studies and future clinical trials.
Considering the suffering of patients with FSHD, the burden of participants is
relatively small and this study can be classified as a study with a negligible
risk. One visit to the LUMC Leiden is required. For collection of blood (DNA,
RNA and plasma), blood samples will be taken through venepuncture, a procedure
with only minor risks. At the site of injection, some redness and swelling may
appear and the individual might experience malaise. For collection of urine,
participants will be asked to collect their urine in a special container. A
skin punch biopsy will be performed, which is minimally invasive and carries
the risk of hematoma and/or local hypoesthesia. Furthermore, a buccal brush
biopsy will be obtained. Participants are given the opportunity to object
against taking a skin punch biopsy or a buccal brush biopsy.