DAG1 haploinsufficiency is associated with sporadic and familial isolated or pauci-symptomatic hyperCKemia

Abstract

DAG1 encodes for dystroglycan, a key component of the dystrophin-glycoprotein complex (DGC) with a pivotal role in skeletal muscle function and maintenance. Biallelic loss-of-function DAG1 variants cause severe muscular dystrophy and muscle-eye-brain disease. A possible contribution of DAG1 deficiency to milder muscular phenotypes has been suggested. We investigated the genetic background of twelve subjects with persistent mild-to-severe hyperCKemia to dissect the role of DAG1 in this condition. Genetic testing was performed through exome sequencing (ES) or custom NGS panels including various genes involved in a spectrum of muscular disorders. Histopathological and Western blot analyses were performed on muscle biopsy samples obtained from three patients. We identified seven novel heterozygous truncating variants in DAG1 segregating with isolated or pauci-symptomatic hyperCKemia in all families. The variants were rare and predicted to lead to nonsense-mediated mRNA decay or the formation of a truncated transcript. In four cases, DAG1 variants were inherited from similarly affected parents. Histopathological analysis revealed a decreased expression of dystroglycan subunits and Western blot confirmed a significantly reduced expression of beta-dystroglycan in muscle samples. This study supports the pathogenic role of DAG1 haploinsufficiency in isolated or pauci-symptomatic hyperCKemia, with implications for clinical management and genetic counseling.

Fig. 1. Summary of genetic findings in heterozygous DAG1 patients.

A Pedigrees of DAG1 families. Loss-of-function DAG1 variants segregate with the hyperCKemia phenotype in all patients. In three families (Family GE-1266_pt1/2, Family GE-184_pt3/4, and Family NA-01_pt8/9), the variants are inherited from a parent presenting with an overlapping clinical phenotype. B Schematic drawing of the dystroglycan protein (NP_001159400.3) showing the localization of the variants identified in our cohort. The variants are scattered across the protein. All the identified DAG1 variants are truncating and predicted to lead to either nonsense-mediated mRNA decay (NMD) or the generation of a truncated transcript. Adapted from ref. [1] (PMID: 16410545).

Fig. 2. Immunohistochemical findings in heterozygous DAG1 patients.

A–C Histopathology studies on muscle biopsy samples from patients #3 and #5. A Hematoxylin and Eosin staining shows mild nonspecific abnormalities, consisting of mild fiber size variability and the presence of internal nuclei in several muscle fibers (white arrows and highlighted in the embedded square box). B Immunofluorescence analysis of members of the dystrophin glycoprotein complex performed on muscle samples of patients #3 and #5 showed significantly reduced signal intensity of beta-dystroglycan protein in both subjects. The differences in the intensity between the two samples may be related to the diverse conditions in which the two pictures were taken and could be influenced by the older age at biopsy in patient #5 compared to #3. C Western Blot analysis probed with anti-beta-dystroglycan on muscle lysates obtained from the same biopsy samples showed a decreased protein expression, with only 43% of normalized expression in both subjects compared to the control.