Molecular Study of the Fukutin-Related Protein (FKRP) Gene in Patients from Southern Italy with Duchenne/Becker-like Phenotype

Abstract

Pathogenic variants localized in the gene coding for the Fukutin-Related Protein (FKRP) are responsible for Limb-Girdle Muscular Dystrophy type 9 (LGMDR9), Congenital Muscular Dystrophies type 1C (MDC1C), Walker-Warburg Syndrome (WWS), and Muscle-Eye-Brain diseases (MEBs). LGMDR9 is the fourth most common hereditary Limb Girdle Muscular Dystrophy in Italy. LGMDR9 patients with severe disease show an overlapping Duchenne/Becker phenotype and may have secondary dystrophin reduction on muscle biopsy. We conducted a molecular analysis of the FKRP gene by direct sequencing in 153 patients from Southern Italy (Calabria) with Duchenne/Becker-like phenotypes without confirmed genetic diagnosis. Mutational screening of the patients (112 men and 41 women, aged between 5 and 84 years), revealed pathogenic variants in 16 subjects. The most frequent variants identified were c.427C > A, p.R143S, and c.826C > A, p.L276I (NM_024301.5). The results obtained show that the Duchenne/Becker-like phenotype is frequently determined by mutations in the FKRP gene in our cohort and highlight the importance of considering LGMDR9 in the differential diagnosis of dystrophinopathies in Calabria. Finally, this study, which, to our knowledge, is the first conducted on Calabrian subjects, will contribute to the rapid identification and management of LGMDR9 patients.

Keywords: FKRP; LGMD2I; LGMDR9; limb girdle muscular dystrophy type R9; molecular dynamic.

Figure 1

Multi-species alignment obtained by UCSC Browser. The symbol R (blue) indicates Arginine in position 143 conserved in Vertebrates.

Figure 2

Three-dimensional structure model of mutated and wild-type FKRP. In green are indicated the amino acid residues of the wild type of Arginine (R) in position 143 and Leucine (L) in position 276 (left panels). In yellow are the variants Serine (S) in position 143 and Isoleucina (I) in position 276 (right panel). The light blue dashed lines indicate the hydrogen bonds, and the orange arrow indicates the new hydrogen bond in the mutated S143.

Figure 3

Three-dimensional surface of the R143S mutated and WT FKRP. Three-dimensional representation of electrostatic (bottom) and hydrophobic (upper) protein surfaces. Left panels show the chain with the Arginine (R) residue in position 143 (WT); right panels show the chain with the Serine (S) variant residue 143. The black closed line indicates the surface subtended by the 143 residues. Color scale is reported on the left side of panels.

Figure 4

Three-dimensional surface of the L276I mutated and WT FKRP. Three-dimensional representation of electrostatic (bottom) and hydrophobic (upper) protein surfaces. Left panels show the chain with the Leucine (L) residue in position 276 (WT); right panels show the chain with the Isoleucine (I) variant residue in position 276. The black closed line indicates the surface subtended by the 276 residues. Color scale is reported on the left side of panels.

Figure 5

GROMACS Backbone Analysis. (A) Root-Mean-Square-Deviation RMSD; (B) Root-Mean-Square-Fluctuation RMSF; (C) Radius of gyration Rg. The values were calculated with respect to the Cα-atom. FKRP WT values are shown in green, while values for the p.R143S and p.L276I variants are shown in red and light blue, respectively. Arrows in (B) indicate some RMSF values of the p.R143S variant clearly different from WT.

Figure 6

PCA of FKRP protein with variants and wild type. Projection of Cα-atoms along the principal two eigenvectors is shown. FKRP protein WT is shown in circle green; FKRP protein with p.R143S and p.L276I variants in square red and diamond light blue, respectively.