Whole exome sequencing identified a novel DAG1 mutation in a patient with rare, mild and late age of onset muscular dystrophy‐dystroglycanopathy

Abstract Muscular dystrophy‐dystroglycanopathy (limb‐girdle), type C, 9 (MDDGC9) is the rarest type of autosomal recessive muscular dystrophies. MDDGC9 is manifested with an early onset in childhood. Patients with MDDGC9 usually identified with defective glycosylation of DAG1, hence it is known as “dystroglycanopathies”. Here, we report a Chinese pedigree presented with mild MDDGC9. The proband is a 64 years old Chinese man. In this family, both the proband and proband's younger brother have been suffering from mild and late onset MDDGC9. Muscle biopsy showed that the left deltoid muscle with an advanced stage of dystrophic change. Immunohistochemistry staining of dystrophin, α‐sarcoglycan, β‐sarcoglycan and dysferlin are normal. Molecular genetic analysis of the proband has been done with whole exome sequencing. A homozygous novel missense mutation (c.2326C>T; p.R776C) in the exon 3 of the DAG1 gene has been identified in the proband. Sanger sequencing revealed that this missense mutation is co‐segregated well among the affected and unaffected (carrier) family members. This mutation is not detected in 200 normal healthy control individuals. This novel homozygous missense mutation (c.2326C>T) causes substitution of arginine by cystine at the position of 776 (p.R776C) which is evolutionarily highly conserved. Immunoblotting studies revealed that a significant reduction of α‐dystroglycan expression in the muscle tissue. The novelty of our study is that it is a first report of DAG1 associated muscular dystrophy‐dystroglycanopathy (limb‐girdle), type C, 9 (MDDGC9) with mild and late age of onset. In Chinese population this is the first report of DAG1 associated MDDGC9.


| INTRODUCTION
Muscular dystrophies are a group inherited disorder characterised by gradual and progressive weakness of muscles. 1 It shows an extreme genotypic and phenotypic heterogeneity. 2 The normal function of human skeletal muscle needs both the intracellular sarcomeric proteins and extracellular matrix (ECM). Intracellular cytoskeleton and ECM are mechanically strongly linked together by a multimeric protein complex named as dystrophin-glycoprotein complex (DGC). 3,4 DGC is composed by intracellular, extracellular and transmembrane proteins. Hence, mutations in any of the proteins of the DGC complex exert recessive negative effects and finally leads to different forms of hereditary muscular dystrophies. 5,6 The dystroglycan (DAG1) gene is located in chromosome 3, comprises of six exons. DAG1 gene encodes dystroglycan protein which is the key component of DGC and plays a significant role in linking dystrophin with other proteins of ECM to form and give mechanical support to the intracellular cytoskeleton. DAG1 gene is evolutionarily highly conserved and dystroglycan is primarily expressed as a precursor protein. During post-translational modification, dystroglycan protein has been cleaved at Ser654 by proteolysis to form α-dystroglycan and β-dystroglycan. 7 In the ECM, the extracellular protein α-dystroglycan is linked with laminin α2. The membrane glycoprotein β-dystroglycan is bind with dystrophin inside the cell and also linked with αdystroglycan extracellularly. Dystrophin is involved in interaction with actin cytoskeleton. Dystroglycans are also involved in signalling pathways by interacting with signalling proteins. 8,9 During post-translational modification, dystroglycan is glycosylated for normal functioning, otherwise it will lose specific interaction with ligands. 4 Till now, mutations of eight human genes are associated with abnormal or defective glycosylation of dystroglycan. 10,11 Recently, they have been collectively designated as "muscular dystrophy-dystroglycanopathies(MDDG)". In addition, MDDG are divided into three types: type A (the most severe, WWS and MEB included); type B (intermediate, congenital muscular dystrophy without brain and eye anomalies included); type C (the mildest, limb-girdle muscular dystrophy included). Besides the deficiency in glycosylation of dystroglycans, a significant reduction in dystroglycan protein level is also evident in several patients with "dystroglycanopathies". 12 Dystroglycan interacts with several intracellular and extracellular proteins or signalling molecules. Intracellularly, β-dystroglycan interacts with dystrophin, utrophin, DRP2 and other signalling molecules by its C-terminal domain. 13 In contrast, extracellularly, αdystroglycan interacts with laminin globular (LG) domains of laminin α2, neurexin, perlecan and agrin by glycosyl residue. 14,15 In our present study, we describe a three generation Chinese family with MDDGC9. Comprehensive clinical evaluation has been done, including muscle biopsy and pathological study. Whole exome sequencing identified a homozygous missense mutation in DAG1 gene in the proband. Immunoblotting study identified that significantly decreased expression of α-dystroglycan in the proband's muscle. DAG1 related muscular dystrophy is very rare and till now only 10 mutations have been reported in DAG1 gene to be associated with muscular dystrophy. So, our present study not only expand the mutational spectrum of the DAG1 gene, but also emphasise the significance of whole exome sequencing for rapid, accurate and cost-effective approach for identifying the novel mutation of candidate genes associated with genetically and phenotypically extremely heterogeneous hereditary muscular dystrophy. This is the first report of DAG1 associated MDDGC9 in China as well as the first report of late age of onset of MDDGC9 worldwide.

| Clinical evaluation
We performed comprehensive clinical examinations related to neuromuscular disorders. We explored routine blood tests including serum creatine kinase level. Electrophysiological study and muscle biopsy were performed. The muscle pathology procedures included standard staining and the immunohistochemistry staining of dystrophin-N, dystrophin-R, dystrophin-C, α-sarcoglycan, β-sarcoglycan and dysferlin-C (mouse monoclonal antibody, Novocastra, US).
F I G U R E 1 Pedigree of the family. The filled symbol indicates the patient (proband), and the half-filled symbols show the carrier parents, who were heterozygous carriers but were unaffected. The arrow points to the proband

| Whole exome sequencing
The genomic DNA was extracted from peripheral blood, randomly fragmented and sheared into fragments of 180-280 bp in length using a Covaris crusher. After the ends were repaired and A tails were added, DNA fragments were ligated to the ends of the fragments to prepare DNA libraries. The exome was enriched using Agilent's SureSelect Human AII Exon V5 Kit, with up to 543872 biotins after library pooling with a specific index. The labelled probes were hybridised in liquid phase. Then 334378 exons of 20965 genes were captured using streptomycin-containing magnetic beads. The libraries were linearly amplified by PCR and subjected to library quality tests.
After passing the test, they were carried out high-throughput deep The detailed variant interpretation process is described in

| Immunoblotting of α-dystroglycan
Muscle sections were prepared and lysed by using lysis buffer and separated by 10% SDS-PAGE followed by transfer of these gels to PVDF membrane. Skimmed milk (5%) was used for blocking the membrane and mouse monoclonal antibody was used to probe αdystroglycan. α-actinin was used as a loading control. Goat antimouse IgG was used to detect the bound primary antibody and performed western blot. 17,18 2.6 | Data availability Nerve conduction velocity and electromyogram tests showed myogenic changes with normal peripheral nerve conduction.

| Pathological result of muscle biopsy
The pathological findings of left deltoid muscle illustrated advanced stage of dystrophic change. There is a predominance of type I fiber. Immunohistochemistry staining of dystrophin, α-sarcoglycan, β-sarcoglycan and dysferlin are normal (Figure 4). In silico analysis was performed to understand and predict the significance of this mutation. In dystroglycan protein, the p.Arg776 is evolutionarily highly conserved among different species ( Figure 6).

| Identification of a novel mutation in DAG1
Hence, we can predict that mutation in this residue can exert a dominant negative effect to the dystroglycan protein structure which renders the normal function of the dystroglycan protein and leads to the disease phenotype.

| Immunoblotting of α-dystroglycan
The α-dystroglycan in the muscle from the patient was identified with significantly reduced expression compared to other control muscle samples (Figure 7). This finding implies the homozygous missense mutation in DAG1 gene impairs the dystroglycan protein structure and function.

| DISCUSSION
Among all the types of inherited muscular dystrophies, MDDGC9 is the rarest form. However, in patients with MDDGC9, defective or abnormal glycosylation or hypoglycosylation of α-dystroglycan leads to the loss of extracellular interaction between α-dystroglycan with laminin which finally results into progressive weakness of skeletal muscle. 5 Here, we identified a three generation Han Chinese family with mild and late onset MDDGC9. Whole exome sequencing and Sanger sequencing identified a novel homozygous DAG1 mutation which is co-segregated well among patients and the unaffected carriers in this family. Functional characterisation identified that this missense mutation causes significant reduction in expression of αdystroglycan compared with control. Hence, we again establish that the important role of α-dystroglycan in normal functioning of the muscle and mutation of it leads to muscular dystrophy. Till date, only 18 genes have been identified to be associated with dystroglycanopathies. [24][25][26][27] Among these 18 genes, germline mutation of DAG1 mutation is associated with primary dystroglycanopathy in limb-girdle muscular dystrophy and muscle-eye-brain disease. 10,11 In addition, due to extreme genotypic and phenotypic heterogeneity, clinical diagnosis through genetic screening for the patients with dystroglycanopathy, is really a big challenge. In this study, we performed whole exome sequencing (WES) for identifying the candidate gene with novel mutation. Our present study strongly emphasises the significance of WES for rapid, accurate and cost-effective approach for identifying the causative gene with pathogenic mutation.
However, DAG1 gene associated MDDGC9 is a very rare dystroglycanopathy and till now less than 10 cases reported worldwide.
Here, the clinical symptoms of patients are also very different from those previous reports as the clinical phenotype of our studied patient is very mild and very slowly progressing with late age of onset. To be the best of our knowledge, this is the first description of a potentially pathogenic mutation of the DAG1 gene associated with MDDGC9 in China.

ACKNOWLEDG EMENT
We are thankful to the proband and all the family members for participating in our study.

CONFLI CT OF INTEREST
The authors confirm that there are no conflicts of interest.