leading FSHD candidate gene, has been shown to activate pathways

involved in sarcomeric protein degradation. Here we investigated whether

sarcomeric dysfunction contributes to muscle weakness, using demembra-

nated single muscle fibers of FSHD patients and control subjects. The force

generating capacity of sarcomeres is significantly impaired in FSHD. Sarco-

meric weakness was restricted to type II muscle fibers, in which maximum

force generation was only 70% of normal strength. In contrast to active

force measurements, a 5- to 12-fold increase in passive force was seen in type

I and type II fibers respectively, indicating stiffening of titin molecules. Cor-

roborating these findings, we observed a decrease in myofilament lattice

spacing and increase in calcium sensitivity, both physiological consequences

of titin stiffening. Based on these findings, we propose that sarcomeric dys-

function plays a critical role in muscle weakness in FSHD.

http://dx.doi:10.1016/j.nmd.2012.06.321

G.P.109

Elucidation of DUX4 binding site affinities by SELEX-Seq

J. Domire 1, S. Coppens 1, L. Wallace 2, S. Guckes 2, S.Q. Harper 1

1 Nationwide Children’s Hospital, Center for Gene Therapy, Columbus,

United States; 2 Nationwide Children’s Hospital, Columbus, United States

Facioscapulohumeral muscular dystrophy (FSHD) is a dominant dis-

order that most commonly affects specific muscles of the face, shoulder gir-

dle, and limbs and has no effective treatment. The relative lack of focus on

targeted FSHD therapies existed because the pathogenic mechanisms

underlying the disease were not well understood. The field may have

reached a tipping point recently with the emergence of a new FSHD path-

ogenesis model involving mis-expression of the pro-apoptotic DUX4 tran-

scription factor. DUX4 belongs to the homeodomain family of proteins

that bind a common AT-rich core motif. Two previous studies reported

DUX4 binding sites using ChIP-seq and EMSA methodology. However,

in our preliminary work using gel shifts we found that the DUX4 protein

was capable of binding numerous homeodomain sites, suggesting the pro-

tein may be “sticky”. We therefore set out to better characterize DUX4

binding sequences and their relative affinities with respect to other double

homeodomain proteins. To do this, we developed a Systematic Evolution

of Ligands by Exponential Enrichment (SELEX) method using human

DUX4 protein. SELEX provides an unbiased method to determine

binding motifs, and has been used to ascertain novel binding sites within

genomes. Briefly, this method involved incubating purified full-length

DUX4 protein with a randomized, 27-mer double-stranded oligonucleo-

tide pool, washing unbound sequences, and PCR-amplifying eluted,

DUX4-enriched binding sites. High-throughput Illumina sequencing in

enriched samples was used to identify DUX4 binding motifs. We are

now using bioinformatic analysis to search for these DUX4 binding sites

in promoters of genes changed in FSHD patient samples and mouse mus-

cles expressing our previously described AAV�DUX4 vectors. This study is

an important early step in identifying DUX4-modulated target genes in

muscle, and may pinpoint relevant pathways for targeted FSHD therapeu-

tics, downstream of DUX4.

http://dx.doi:10.1016/j.nmd.2012.06.322

G.P.110

The DUX4 promoter is preferentially expressed in FSHD-affected tissues

L. Wallace, J. Liu, S. Harper

Nationwide Children’s Hospital, Gene Therapy, Columbus, United States

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal

dominant disorder with a one in 7500 incidence. Symptoms typically arise

in young adulthood and most patients show clinical features before age 30.

FSHD is most commonly characterized by progressive wasting and weak-

ness of facial and shoulder-girdle muscles, although no consistent pattern

of penetrance or severity exists, even within affected families. The hallmark

characteristic of FSHD is asymmetrical muscle weakness. There are also

non-muscular features including retinal vasculopathy and high frequency

hearing loss. The pathogenic mechanisms underlying FSHD have been dif-

ficult to discern, and scientists in the field have spent most of the last two

decades attempting to decipher FSHD pathogenesis. Several recent break-

throughs now support a model in which mis-expression of the myotoxic

DUX4 gene is a primary pathogenic event underlying FSHD. We recently

showed that DUX4 is generally toxic to muscles, and we therefore hypoth-

esized that there was a direct correlation between DUX4 expression pat-

terns and the involvement of only selected muscles (and ear and retinal

pathologies) in FSHD. In short, we proposed that if DUX4 over-expres-

sion is indeed the underlying pathogenic event in FSHD, it must be pref-

erentially expressed in FSHD-affected regions. To test this hypothesis, we

developed transgenic reporter mice containing a putative DUX4 promoter

cloned upstream of GFP. We generated three separate lines of DUX4

promoter-GFP mice. We found the DUX4 promoter directed GFP

expression in the face and limbs of newborn and adult mice, as well as

the retina and inner ear. Essentially all other organs were GFP negative.

Strikingly, all lines showed asymmetrical expression and variable

penetrance, even within individual litters. We conclude that our mice faith-

fully recapitulate expected DUX4 expression patterns in regions of FSHD

pathology, and further support the role of DUX4 as pathogenic insult in

FSHD.

http://dx.doi:10.1016/j.nmd.2012.06.323

G.P.111

Exudative retinopathy in facioscapulohumeral muscular dystrophy: When to

screen? When to monitor? When to treat?

R.B. Fitzsimons

University of Sydney, Sydney, Australia

Visual loss caused by exudative retinal detachment is a rare but pre-

ventable association of FSHD. Underlying peripheral retinal vascular tel-

angiectasis is however very common. Focal retinal hypovascularity may be

a developmental abnormality parallel to aberrations in myogenesis. Since

asymptomatic telangiectasis appears likely to remain static for many years,

the question arises as to proper practice in relation to indirect ophthalmo-

scopic screening of FSHD patients, particularly children, and if and when

to treat visualized exudates, which may track from the retinal periphery to

the posterior pole. Accumulated international experience has demon-

strated the efficacy of unusually aggressive and frequent treatment, typi-

cally with Laser photocoagulation directed at leaking vessels, even when

vision is significantly impaired. However, once macular damage is estab-

lished, visual loss may be irreversible. In advanced stages, painful glau-

coma may lead to enucleation. Family follow-up now underlines the

particular importance of reviewing patients from FSHD families with a

history of overt retinal vascular disease. Two members of one family lost

vision unilaterally in mid-life; only one seen early enough to treat success-

fully. Retinal abnormalities are (like the myopathy) asymmetrical; bilat-

eral visual loss is rare but real. It is concluded that (i) severe and early-

onset FSHD patients with clinical deafness are more likely to have treat-

able retinal exudates, and should be monitored closely, especially in early

childhood. (ii) Retinal disease may present before muscle disease. (iii)

Atypical cases of Coats Disease (e.g. when bilateral, in women, or in older

patients) may justify genetic testing, with caution, for FSHD. (iv) All

FSHD patients should have one indirect ophthalmoscopy examination

and report unexplained visual symptoms immediately. (v) Peripheral exu-

Abstracts / Neuromuscular Disorders 22 (2012) 804–908 901