Molecular Diagnosis In An Autosomal Recessive Family With Neuromuscular Disease

There are many kinds of inherited neuropathy diseases, which have complex pathogenesis and mechanism. The characteristics of inherited neuropathies are familial and lifelong. The rate of malformation and disability of the diseases is very high. There is no effective way to cure inherited neuropathies so far, so it is a great burden for the families and society. In recent years, the pathogenesis and molecular mechanism of inherited neuropathies are gradually clear, due to the development of genetics and molecular biology.This will provide a significant help for genetic diagnosis, genetic counseling and prenatal diagnosis.Limb girdle muscular dystrophy is a group of hereditary neuromuscular disease with a high degree of genetic heterogeneity and clinical heterogeneity, which is characterized by proximal weakness with sparing of the facial and distal muscles, although distal weakness may occur later in the course or in severe cases. Adolescent onset and the main clinical features contain limb proximal muscle weakness and atrophy, with or without muscle biopsy showing rimmed vacuoles. According to the inheritance pattern, LGMD can be divided into LGMDl （autosomal dominant） and LGMD2 （autosomal recessive）. There are 7 subtypes of LGMD1 （1A-1G）, while LGMD2 has 23 subtypes （2A-2W）, according to the pathogenic genes. More than 90% of LGMD are autosomal recessive. A muscle fiber protein complex is composed of limb muscular dystrophy protein and the Dystrophin-glycoprotein complex, which is attached to the muscle fiber membrane. Close connection exists between the proteins and deletion or mutations of any protein will affect the stability of the entire structure of the membrane, resulting in muscle cell necrosis. LGMD2G is relatively rare, only about 20 cases of families have been reported worldwide.Hereditary inclusion body myopathy （h-IBM）, late adolescence or early adult onset, its main pathological changes are skeletal muscle fiber atrophy and necrosis, containing aggregates of inclusion bodies rimmed vacuoles. Distal myopathy rimmed vacuoles （DMRV） is the most common form of h-IBM, mainly showing atrophy of the distal muscle weakness and the latter it can affect the proximal muscles. The lower limb and anterior tibial muscle are most seriously affected, while the quadriceps femoris is less affected. The upper limb to shoulder muscles is also severely defected. DMRV is tied to mutations of GNE and more than 60 mutations have been verified.Diagnosis of hereditary neuromuscular disorders mainly depends on clinical symptoms, genetic pattern, serum CK and muscle pathology many years ago. The clinical features of LGMD2G and DMRV were very similar, such as autosomal recessive inheritance, progressively dystrophies of limb-girdle muscular and high level of serum creatine-kinase （CK）. Part of the LGMD2G patients have a large number of muscle fibers with rimmed vacuoles and some of the DMRV patients’ proximal limb muscle is affected. Therefore, it is difficult to make a definite diagnosis only simply based on the clinical features of patients, so molecular diagnosis becomes a gold standard for the diagnosisIn this study, we have collected a consanguineous family with neuromuscular disease from Shandong province. The medical history and the clinical symptoms of the patient were recorded. Physical examinations and other essential auxiliary examinations were carried out. The patient has undergone progressive muscular weakness for 5 years. Imaging examinations （including lower limbs MRI, lower limbs CT three-dimensional imaging） indicated muscular atrophy of lower limbs. Biopsy of anterior tibialis showed the muscular fibers were in different sizes. The muscular fibers have massive cystic vesicles within and are myogenic damaged. Blood CK level raises apparently. The pedigree is shown as autosomal recessive inheritance pattern. According to the characteristics above, we tentatively diagnosed the patient as distal myopathy with rimmed vacuoles （DMRV）. Because the only pathogenic gene of DMRV is known as GNE so far, we first detected the total coding region of GNE in the proband by Sanger sequencing. However, no mutation was found in the affected individuals. We carried out homozygous mapping of all members of the pedigree.7 positive regions were found by homozygous mapping. Then we conducted whole-exome sequencing for the proband and filtered the data by dbSNP,1000 genomes. There were 195 mutations remained at last. After analyzing the mutations located in homozygous positive regions, we targeted the pathogenic gene as TCAP. Patients carried homozygous mutation of TCAP （c.165₁66insG） and this mutation was not been found in Exome Variant Server （EVS） and Exome Aggregation Consortium （ExAC） database. In order to confirm that TCAP is the pathogenic gene of this pedigree, we constructed wild type and mutant type expression vectors of TCAP, and screened a stable cell line, HEK293. Based on Western blotting results, we discovered the mutant protein of TCAP was a truncating mutation and the expression of mutant protein is in a low level. Above all, it suggested that mutant TCAP protein was severely impaired. According to pathogenic gene and functional analysis of mutant gene, we confirmed that TCAP was the pathogenic gene of this pedigree. It has been reported that TCAP is the pathogenic gene for limb-girdle muscular dystrophy. Moreover, DMRV and LGMD2G have a similar phenotype, and it is difficult to distinguish them according to clinical features and common auxiliary examinations. We ultimately diagnosed the patient as limb-girdle muscular dystrophy type 2G based on both pathogenic gene and clinical features.