G.P.149

Unique brick-red auto-fluorescence of reducing bodies and protein aggre-

gates is a useful diagnostic biopsy marker for FHL1-associated myopathies

R. Phadke

Dubowitz Neuromuscular Centre, UCL Institute of Neurology, London,

UK

Mutations in the four and a half LIM domain protein 1 (FHL1) gene

are causative of four distinct human myopathies with a wide

clinicopathological spectrum. A biopsy diagnosis can be difficult due to

overlap with other protein aggregation myopathies and dystrophic

changes. The main aim was to assess the auto-fluorescent characteristics

of inclusions in biopsies from molecularly confirmed cases of FHL1 and

compare them with other protein aggregation myopathies. 14 muscle

biopsies taken from patients referred to our neuromuscular centre were

reviewed retrospectively (FHL1 = 4, Bag3 = 3, Desmin = 1,

Myotilin = 1, Acid maltase = 1, Colchicine myopathy = 1, Autophagic

vacuolar myopathy = 1, Inclusion body myositis = 2). Frozen sections

stained with haematoxylin and eosin were assessed with bright field

microscopy and fluorescent microscopy using BGR multi-excitation

wavelength filter on a Leica DM2500 microscope except in one FHL1

case where paraffin sections were examined. Relevant histochemistry,

immunostains and electron microscopy were also assessed. Unique

brick-red auto-fluorescence of the inclusions including discrete reducing

bodies and majority of diffuse protein aggregates was seen only in

frozen sections of FHL1 cases. Non-overlapping but closely interacting

brick-red auto-fluorescence of FHL1 inclusions with yellow-orange

autofluorescence of desmin and myotilin was also seen. Red

auto-fluorescent material was present in and around degenerate

myonuclei. Reducing bodies and diffuse reducing body material was

confirmed electron microscopically. Protein aggregates in all other

non-FHL1 cases showed variable yellow to orange auto-fluorescence.

Menadione-NBT reduction without substrate was also seen in

non-FHL1 protein aggregates. Brick-red auto-fluorescence appears to be

a unique property of FHL1 containing reducing bodies and protein

aggregates and may be a useful biopsy marker for the diagnosis of

FHL1 myopathies.

http://dx.doi:10.1016/j.nmd.2014.06.179

G.P.150

Clinical heterogeneity in adult forms of FHL1 related myopathies. The

“Institut de Myologie” experience

R.A.B. Ben Yaou 1, T.A.N. Stojkovic 1, P.A.S. Laforet 1,

A.L.I. De Becdelievre 2, H.E.N. Becane 1, K.A.R. Wahbi 1,

C.A.R. Navarro 3, M.I.C. Fardeau 1, N.O.R. Romero 1, P.A.S. Richard 4,

D.E.N. Duboc 5, G.I.S. Bonne 1, B.R.U. Eymard 1

1 Institut de myologie, APHP, GH Pitie Salpetriere, Paris,

France; 2 Institut de myologie, GH Pitie Salpetriere, Paris,

France; 3 Institute of Biomedical Research of Vigo (IBIV), University

Hospital of Vigo (CHUVI), Paris, France; 4 APHP, GH Pitie Salpetriere,

Paris, France; 5 APHP, GH Cochin, Paris, France

FHL1 gene mutations are responsible for reducing body myopathy

(RBM), a rare condition characterized by progressive muscle weakness

and the presence of intracytoplasmic aggregates. Age at onset ranges

from early onset in infancy, through childhood and in some cases adult

age. FHL1 mutations may also lead to allelic disorders including

Emery-Dreifuss like muscular dystrophy (EDMD), hypertrophic

cardiomyopathy (HCM), X-linked myopathy with postural muscle

atrophy and generalized hypertrophy (X-MPMA) and X-linked

scapuloperoneal myopathy (X-SM). To report clinical, muscle imaging,

histological and genetic features found of adult patients carrying FHL1

mutations, we retrospectively reviewed their medical reports of the 11

patients (6 M, 5 F) belonging to 8 families followed at the “Institute de

Myologie”, Pitie-Salpetriere hospital, Paris. The 11 patients were

assessed from 13 to 52 years old. Pseudodominant mode of inheritance

was suggested in 4 families, X-linked in 3 and sporadic in one. Four

had EDMD, 2 X-SM, 1 X-MPMA, 1 HCM while 2 patients had

atypical features and 1 was still asymptomatic. When symptomatic,

female patients had a striking asymmetric muscle involvement. Five

patients had hypertrophic cardiomyopathy requiring heart

transplantation in one patient at 18 years old. Two patients required

night time ventilation due to diaphragm paralysis. Histologically, only 3

patients among the 8 probands had reducing bodies (RBs) at muscle

biopsy, while 3 had non specific pattern, 1 had neuropathic pattern and

one muscle biopsy was considered as normal. In contrast to infantile

forms of FHL1 related myopathy that usually show RBs at muscle

biopsy and lead to early death, adult forms had wider clinical and

histological presentations. Moreover, FHL1 gene screening should be

considered in those undiagnosed patients suffering from myopathies

with diaphragm paralysis and/or HCM where X-linked inheritance is

not excluded even in the absence of RBs at muscle pathology.

http://dx.doi:10.1016/j.nmd.2014.06.180

G.P.151

Loss of FHL1 function impairs motility and causes myopathy in vivo

M. Keßler 1, A. Kieltsch 1, E. Kayvanpour 2, B. Schoser 3, J. Schessl 3,

W. Rottbauer 1, S. Just 1

1 Universitatsklinikum Ulm, Ulm, Germany; 2 Universitatsklinikum

Heidelberg, Heidelberg, Germany; 3 Universitatsklinikum Munchen,

Munchen, Germany

Mutations of the X-chromosomal four and a half LIM domain 1

(FHL1) lead to inclusion body myopathies such as reducing body

myopathy or Emery-Dreifuss muscular dystrophy, termed as

FHL1-opathies. Family screens identified association of different

missense mutations of FHL1 to skeletal muscle myopathy either isolated

or linked with cardiomyopathy. Until now it is unknown whether

dominant negative effects (accumulation of cytotoxic mutated FHL1

protein) or loss of FHL1 function (reduced FHL1 protein levels)

underlie the pathogenesis of these FHL1-opathies. Therefore, we

exploited the model organism zebrafish to evaluate the role of FHL1 for

pathogenesis of FHL1-opathies in vivo. By Morpholino-modified

oligonucleotide mediated gene knockdown of the two zebrafish FHL1

orthologs, fhl1a and fhl1b, we examined loss of FHL1 function in vivo.

In both morphant embryos we find impaired motility and skeletal

muscle function consistent with FHL1-opathy. To evaluate whether

three common missense FHL1-mutations identified in FHL1-opathy

affected patients either act dominant-negative or lead to loss of FHL1

function in vivo, we injected mutated human FHL1 mRNA in both

wild-type and fhl1-depleted zebrafish embryos. Hence, injection of

mutated human FHL1 mRNA into wild-type embryos, does not impact

on skeletal muscle function, suggesting that the investigated mutations

do not act dominant-negative in vivo. Interestingly, in contrast to

wild-type FHL1 mRNA, expression of the mutated human FHL1 in

fhl1-depleted embryos was not capable of reconstituting skeletal muscle

function, indicating that the common human FHL1 mutations lead to a

loss of FHL1 function thereby causing the FHL1-opathy. In summary,

we demonstrate here, that loss of FHL1 function leads to impaired

motility and myopathy in the zebrafish in vivo model. Furthermore, our

functional studies reveal that three common human FHL1-mutations do

not act dominant-negative in vivo but rather lead to a loss of FHL1

function.

http://dx.doi:10.1016/j.nmd.2014.06.181

846 Abstracts / Neuromuscular Disorders 24 (2014) 791–924