Mild limb girdle muscular dystrophy R9 phenotype caused by novel compound heterozygous FKRP gene mutation

Abstract

Fukutin-related protein (FKRP) mutations cause a broad spectrum of muscular dystrophies, from a relatively mild limb-girdle muscular dystrophy type 9 (LGMDR9) to severe congenital muscular dystrophy (CMD). This study aims to report two siblings belonging to a non-consanguineous Tunisian family harboring a novel compound heterozygous FKRP variant and presenting a mild LGDMR9 phenotype. For mutation screening, massive parallel sequencing was performed, followed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to validate the existence of the discovered variants. The absence of alpha-dystroglycan was determined by immunohistochemistry. Brain and thigh magnetic resonance imaging (MRI) were performed to detect thigh and brain abnormalities. The two siblings had a late age at onset and clinical examination showed that the pelvic girdles had a predominantly proximal and symmetrical distribution of weakness without cardiac or respiratory involvement. They both had a modified Gardner-Medwin Walton Scale mGMWS grade of 4 and a modified Rankin Scale (mRS) score of 1. The DNA sequencing revealed a novel deletion of exons 2 and 3 in one allele and a missense mutation c.1364C > A, which has been reported to be responsible for congenital muscular dystrophy and mental retardation on the second allele. The simultaneous presence of the two variations in the two cases suggests that the variants segregate with the pathophysiology.

Keywords: Dystroglycanopathy; FKRP; LGMDR9; limb girdle muscular dystrophy; α-Dystroglycan.

Figure 1.

Histopathology and Immunohistochemical staining of deltoid muscle biopsy. (A) Hematoxilin phloxine saffron staining showing a great fibers size variability with rounded atrophic fibers and hypertrophic fibers. Somme fibers are necrotic undergoing macrophagic resorption (arrow), other fibers present rimmed vacuoles (asterix). (B) Gömöri trichrome stain revealing the presence of rimmed vacuoles and segmentation of many fibers. Immunohistochemical reaction showing normal expression of utrophine (C) and dystrophin (D) and a total absence of α-DG (E). (F) positive control showing a normal presence of α-DG in a muscle biopsy.

Figure 2.

Sanger electropherogram of the proband 1, the proband 2 and the mother samples shows the substitution of cystosine by adnenin in a heterozygous form at the position 1369. The electropherogram of the father sample shows the absenc e of this substitution and the presence of cytosine in a homozygous form at the position 1369.

Figure 3.

Ratio chart produced using MLPA analysis software Coffalyser.net. A probe ratio below 0.7 indicates a heterozygous deletion,whereas a ratio of 1 indicates a normal copy number. The ratio chart of the proband 1 (A), the proband 2 (B) and the father (C) shows 2 consecutive data points (shown in yellow) below the 0.7 ratio mark, indicating the deletion of the two exons 2 and 3 in these samples. (D) Ratio chart of the mother sample showing the absence of data point below 0.7 ratio mark which indicates the absence of the deletion of exon 2 and 3.

Figure 4.

Ratio values of the proband 1 sample (A, proband 2 sample (B), the father sample (C) and the mother sample (D) generated by coffalyser software.

Figure 5.

Proband 1 cerebral and thigh MRI. (A) Axial T1 weighted thigh MRI. (B) Axial T2-weighted sequences of brain MRI (C) Sagittal T1 weighted brain MRI.