PMC9911805_01
Family tree. Family tree showing that the proband (IV-1) and his affected sister (IV-2) were born to consanguineous first cousin parents (III-1 and III-2). Two sisters in their 60s were unaffected (IV-3 and IV-4). Square, male; circle, female; black circle or square, clinically affected; white circle or square, clinically unaffected; line through circle or square, deceased (age of death, where known, given below circle or square as denoted by d.); double line, consanguinity; small black arrow, proband.
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F1
The male proband (IV-1) was the first child of consanguineous first cousin parents (III-1 and III-2) (Figure 1).
MRI brain of proband. Axial FLAIR* image from MRI brain of proband at 64 years of age showing confluent, bilateral white matter T2# hyper intensity with involvement of the corpus callosum *FLAIR, Fluid-attenuated inversion recovery; #T2, transverse relaxation time.
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F2
T2 hyperintensity with involvement of the corpus callosum (Figure 2). There was no significant cerebral or hippocampal atrophy.
PMC9911805_02
Histology, immunohistochemistry and Western blot analysis. (A) Haematoxylin and eosin (H&E) staining of muscle biopsies obtained from the proband (P) at age 56 years (i; quadriceps) and 64 years (ii; biceps). Unstained areas observed in (i) represent adipose tissue infiltrates while muscle fibers appear pink in H&E-stained sections. (B) Merosin immunohistochemical staining of proband (P) quadriceps muscle biopsies and a muscle biopsy obtained from the proband's sister (S) at 59 years demonstrating membrane-staining comparable to control 1 (C1). Immunostaining for spectrin, another membrane-associated protein, showed a staining pattern similar to merosin. (C) Immunohistochemical staining of proband biceps muscle obtained at age 64 years with two merosin antibodies (i: Chemicon MAb1922; ii: NCL-merosin Novocastra) demonstrated membrane-associated staining pattern of merosin similar to (B). (D) Western blot of muscle from the proband (P; 56y) and his sister (S; 59y) showed merosin protein levels similar to those detected in three control samples: C1 = 56y (female), C2 = 57y (female), C3 = 59y (male). (E) Bar graphs of merosin protein levels normalized to actin (measured from total protein stain) (i), dystrophin protein levels normalized to total protein (ii) and merosin protein levels normalized to dystrophin (iii). Average +- standard deviation for proband and sister (collectively denoted as P) and controls (C) are shown. Groups were compared by using a t-test and found to be not significantly different (ns, i; p = 0.4848, ii; 0.0734, iii; 0.1926).
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F3
A quadriceps muscle biopsy obtained from the proband at age 56 years showed myopathic changes with marked replacement of muscle with adipose tissue (Figure 3Ai). A biceps muscle biopsy taken subsequently, aged 64 years, showed less severe myopathic changes compared to his quadriceps biopsy with increased internal nuclei, variation in myofibre diameters including hypertrophic and atrophic fibers, but no adipose tissue infiltration (Figure 3Aii). Merosin immunohistochemistry on two biopsies from the proband (age 56 and 64 years) and one biopsy from the sister (age 59 years) demonstrated membrane-associated staining pattern comparable to control muscle (Figures 3B, C). Merosin protein levels as determined by Western blot were also comparable to controls (three age matched controls were used) in both subjects (Figure 3D). Merosin protein levels were found to be lower in the proband and sister compared to controls when normalized to actin and dystrophin but this difference was not statistically significant (Figure 3Ei, iii). There was a small increase in dystrophin protein levels when normalized to total protein in the proband and sister compared to controls but this difference was also not statistically significant (Figure 3Eii).