Clinical, histological and genetic characterization of reducing body myopathy caused by mutations in FHL1

Abstract

We recently identified the X-chromosomal four and a half LIM domain gene FHL1 as the causative gene for reducing body myopathy, a disorder characterized by progressive weakness and intracytoplasmic aggregates in muscle that exert reducing activity on menadione nitro-blue-tetrazolium (NBT). The mutations detected in FHL1 affected highly conserved zinc coordinating residues within the second LIM domain and lead to the formation of aggregates when transfected into cells. Our aim was to define the clinical and morphological phenotype of this myopathy and to assess the mutational spectrum of FHL1 mutations in reducing body myopathy in a larger cohort of patients. Patients were ascertained via the detection of reducing bodies in muscle biopsy sections stained with menadione-NBT followed by clinical, histological, ultrastructural and molecular genetic analysis. A total of 11 patients from nine families were included in this study, including seven sporadic patients with early childhood onset disease and four familial cases with later onset. Weakness in all patients was progressive, sometimes rapidly so. Respiratory failure was common and scoliosis and spinal rigidity were significant in some of the patients. Analysis of muscle biopsies confirmed the presence of aggregates of FHL1 positive material in all biopsies. In two patients in whom sequential biopsies were available the aggregate load in muscle sections appeared to increase over time. Ultrastructural analysis revealed that cytoplasmic bodies were regularly seen in conjunction with the reducing bodies. The mutations detected were exclusive to the second LIM domain of FHL1 and were found in both sporadic as well as familial cases of reducing body myopathy. Six of the nine mutations affected the crucial zinc coordinating residue histidine 123. All mutations in this residue were de novo and were associated with a severe clinical course, in particular in one male patient (H123Q). Mutations in the zinc coordinating residue cysteine 153 were associated with a milder phenotype and were seen in the familial cases in which the boys were still more severely affected compared to their mothers. We expect the mild end of the spectrum to significantly expand in the future. On the severe end of the spectrum we define reducing body myopathy as a progressive disease with early, but not necessarily congenital onset, distinguishing this condition from the classic essentially non-progressive congenital myopathies.

Fig. 1

Clinical picture. (A) Patient 1, at 8 years of age is permanently wheelchair dependent, she is ventilated via tracheostomy and BiPAP and all nutrition and fluids are provided via gastrostomy tube. Her antigravity strength is limited to her finger extensors and toes. There is facial weakness and asymmetrical ptosis. (B) The male Patient 6 at age 2 years is completely paralysed requiring ventilation via tracheotomy and nutrition via G-tube. Note foot contractures. (C and D) Familial Patient 8 demonstrating scoliosis, spinal rigidity and contractures of knees and elbows in a pattern reminiscent of the Emery-Dreifuss phenotype. (E) Patient 3 showing early prominent periscapular involvement leading to scapular winging.

Fig. 2

Histological appearance. (A and B) Menadione-NBT staining of normal muscle (A) and the muscle biopsy of Patient 1 with evident reactive intracytoplasmic aggregates (arrows) (B). (C–H) Haematoxylin and eosin staining demonstrates darkly staining aggregates (arrows), note degenerating fibre loaded with inclusions (short arrows) (C); modified Gomori trichrome demonstrates the dark red staining aggregates (arrows) contained in a subset of fibres (inset demonstrates a fibre with rimmed vacuole, arrows) (D). NADH (E), and ATPase at pH 9.4 (F), demonstrate that the aggregates are negative on these stains (arrows), but are positive on Congo red stain (arrows) (G). Semi thin section (H) shows characteristic aggregates (arrows) in a longitudinal section.

Fig. 3

Immunohistochemical analysis. (A) Serial sections stained with menadione-NBT and immunostained with anti-FHL1 antibody (directed against a unique amino acid sequence located in the fourth LIM domain of the human FHL1, Monash University, Australia) demonstrating that the menadione-NBT positive aggregates are also FHL1 immunoreactive (arrows). (B and C) FHL1 immunostaining on sequential biopsies from the quadriceps from Patient 6, taken at 2 years of age (B) and 2¼ years of age (C), demonstrate an increase of FHL1 positive aggregates (arrows) (even allowing for the relatively poor preservation of the first biopsy material). (D–F) FHL1 positive aggregates in RBM are also immunoreactive for the centrosomal component pericentrin (D) as well as for gamma-tubulin (F). Gamma-tubulin and pericentrin co-localize for most, but not all aggregates (E).

Fig. 4

Ultrastructural analysis. (A–C) Localization of aggregates (arrows) in close proximity to a nucleus (star), appearing to indent the nucleus (A). Dense material is seen surrounding the myonucleus (star) as a thin band (B). (D and E) Cytoplasmic bodies (arrows) were found in frequent coexistence with the reducing bodies in all specimens examined. (F) Rimmed vacuoles (arrows) occur in some fibres in RBM (compare Fig. 1D inset). (G) Immuno EM: gold-particles indicate binding of the FHL1 antibody to the major electron-dense aggregates.

Fig. 5

Genetic analysis. (A) Pedigrees of families with affected members, indicating mutations in FHL1. Solid symbols designate affected individuals carrying the mutation indicated. Open symbols indicate unaffected individuals not carrying the mutation. (B) Schematic representation of the domain structure of FHL1, comprised of four LIM domains (LIM1-4) with an additional N-terminal half-LIM domain (Z). The secondary structure of the second LIM domain is indicated with the Zn coordinating residues highlighted in bold, and mutated residues in addition in red. Histidine 123 is mutated independently in six patients.