Clinical, genetic, and pathologic characterization of FKRP Mexican founder mutation c.1387A>G

Abstract

Objective: To characterize the clinical phenotype, genetic origin, and muscle pathology of patients with the FKRP c.1387A>G mutation.

Methods: Standardized clinical data were collected for all patients known to the authors with c.1387A>G mutations in FKRP. Muscle biopsies were reviewed and used for histopathology, immunostaining, Western blotting, and DNA extraction. Genetic analysis was performed on extracted DNA.

Results: We report the clinical phenotypes of 6 patients homozygous for the c.1387A>G mutation in FKRP. Onset of symptoms was <2 years, and 5 of the 6 patients never learned to walk. Brain MRIs were normal. Cognition was normal to mildly impaired. Microarray analysis of 5 homozygous FKRP c.1387A>G patients revealed a 500-kb region of shared homozygosity at 19q13.32, including FKRP. All 4 muscle biopsies available for review showed end-stage dystrophic pathology, near absence of glycosylated α-dystroglycan (α-DG) by immunofluorescence, and reduced molecular weight of α-DG compared with controls and patients with homozygous FKRP c.826C>A limb-girdle muscular dystrophy.

Conclusions: The clinical features and muscle pathology in these newly reported patients homozygous for FKRP c.1387A>G confirm that this mutation causes congenital muscular dystrophy. The clinical severity might be explained by the greater reduction in α-DG glycosylation compared with that seen with the c.826C>A mutation. The shared region of homozygosity at 19q13.32 indicates that FKRP c.1387A>G is a founder mutation with an estimated age of 60 generations (∼1,200-1,500 years).

Figure 1. Comparative ancestry and FKRP haplotype sharing

(A) Map of reported family origins of patients homozygous for FKRP c.1387A>G. Blue markers represent patients 2, 3 (siblings), and 6, pink markers represent patient 9's distant grandparents, and purple markers represent 3 previously reported homozygous FKRP c.1387A>G cases. (B) Global ancestry proportions estimated with ADMIXTURE (K = 3) for FKRP patients 0, 3, 4, 5, 6, 9, A, B, and C, compared with 1000 Genomes Project samples from unrelated Native Americans (MXL, 34 samples; PEL 20 samples; CLM 20 samples), Europeans (IBS, 20 samples; CEU 20 samples), and African Americans (ASW, 20 samples). Continental ancestry fraction is shown as Native American (red), European (orange), and African (blue). (C) Heterozygosity for 701 SNPs from chr19:46,664,561-47,933,257 (hg19), with shared homozygous regions for c.1387A>G highlighted in blue and c.826C>A in green. (D) Phased haplotypes from patient 9 (heterozygous c.1387A>G/c.826C>A), patient 5 (c.1387A>G), and patient B (c.826C>A) with red/gray indicating the allele at each SNP position and the minimally shared homozygous regions highlighted in blue/green. SNP = single nucleotide polymorphism.

Figure 2. Histopathology

(A) Representative image of muscle biopsy from a patient homozygous for the European common mutation in FKRP c.826C>A (patient D) showing mild to moderate dystrophic changes. (B) Representative image of muscle biopsy from patient 9 (heterozygous for c.1387A>G and c.826C>A) showing similar changes to the biopsy in part A. (C and D) Representative images from patients 3 and 4 (both homozygous for c.1387A>G) showing a very severe dystrophic, nearly end-stage histopathology. Scale bar = 100 μm, equivalent for all photomicrographs.

Figure 3. Immunofluorescence staining

(A–D) Normal control muscle staining patterns for each of the antibodies. (E–H) Representative images from patient D homozygous for the European common mutation (c.826C>A). (I–L) Representative images from patient 3 homozygous for c.1387A>G. (M–P) Representative images from patient 4 homozygous for c.1387A>G. Scale bar = 50 μm, equivalent for all photomicrographs.

Figure 4. Western blotting

The antipeptide antibody (AF6868) shows greatly reduced molecular weight for α-DG in each of the patients with FKRP mutations. Fully glycosylated control (C) α-DG is >150 kd, whereas the α-DG from homozygous c.1387A>G patients (3 and 5) ranges from ∼65–90 kd, and the α-DG from homozygous c.826C>A (D) or compound heterozygous c.1387A>G/c.826C>A (9) patients ranges from ∼75–90 kd. The smaller molecular weight α-DG observed in homozygous c.1387A>G patients suggests a greater degree of hypoglycosylation than that of c.826C>A patients. Each patient with FKRP mutations has lost functional glycosylation and no longer binds the anti-glycoepitope antibody (IIH6). The AF6868 antibody binds to epitopes on both α-DG and β-DG. The β-DG bands show the relative amounts of protein loaded in each lane. Lanes were loaded equivalently in both gels. These images are representative of blots performed 3 or 4 times for each patient sample.