Novel diagnostic features of dysferlinopathies

Abstract

Reports of dysferlinopathy have suggested a clinically heterogeneous group of patients. We identified specific novel molecular and phenotypic features that help distinguish dysferlinopathies from other forms of limb-girdle muscular dystrophy (LGMD). A detailed history, physical exam, and protein and mutation analysis of genomic DNA was done for all subjects. Five of 21 confirmed DYSF gene mutations were not previously reported. A distinct "bulge" of the deltoid muscle in combination with other findings was a striking feature in all patients. Six subjects had atypical calf enlargement, and 3 of these exhibited a paradoxical pattern of dysferlin expression: severely reduced by direct immunofluorescence with overexpression on Western blots. Six patients showed amyloid deposits in muscle that extended these findings to new domains of the dysferlin gene, including the C2G domain. Correlative studies showed colocalization of amyloid with deposition of dysferlin. The present data further serve to guide clinicians facing the expensive task of molecular characterization of patients with an LGMD phenotype.

Figure 1

Localization of the mutations in the dysferlin gene and the corresponding exons and protein domains. A green background color was used to distinguish the patients with amyloid deposits in their muscle biopsy. The number inside the box corresponds to the number of the patient in Table 2.

Figure 2

(A-F) Shows deltoid hypertrophy in 6 cases of dysferlin deficiency. The surrounding muscles are atrophic. This is well seen in (A) and (B), where the atrophy of the biceps is clearly demonstrated.

Figure 3

(A,B) Calf muscle hypertrophy in patients with dysferlin gene mutations. These patients had been previously mislabeled with a diagnosis of Becker muscular dystrophy.

Figure 4

(A) Congo red stain shows amyloid deposited in the smooth muscle layer of a skeletal muscle arteriole in patient 3. (B) Using polarized light, the green birefringence of amyloid is seen. (C) In a consecutive serial section, dysferlin deposition is seen in this same blood vessel (NCL-Hamlet Novocastra Laboratories Ltd.). (D) In the same section a blood vessel wall lacks dysferlin deposition; only the autofluorescence of the internal elastic membrane (tunica intima) and adventitia (tunica externa) can be seen, but the smooth muscle of the tunica media is devoid of dysferlin.

Figure 5

(A) Dysferlin staining in the sarcolemma of normal muscle; (B) negative control (normal muscle without dysferlin antibody stained only with goat anti-mouse IgG); (C) reduced dysferlin membrane staining is seen in patient 2 (Table 2); (D) absent dysferlin membrane staining in patient 8 (Table 2). NCL-Hamlet antibody Novocastra Laboratories Ltd in A,C, D.

Figure 6

Western blot in LGMD2B patients. Total protein extracted from the patient muscle samples was probed with dysferlin antibody (upper panel). Lane 1: a normal control; lanes 2-4: LGMD2B patients showing overexpression of dysferlin in patients 3, 8, 10, Table 2); band signal intensities were quantified by ImageQuant T2 software (GE Healthcare) and were to 2 to 5 fold increased over normal controls; lanes 5-8: LGMD2B patients showing no dysferlin expression. Protein loading is assessed by probing with muscle specific actin antibody (lower panel).