A novel SYNE1 gene mutation in a Chinese family of Emery-Dreifuss muscular dystrophy-like

A total of six individuals from a family of four generations were assessed (Fig. 1), alongside 100 unrelated normal controls matched for age and gender. All participants provided informed consent before enrolment. The experimental protocol was approved by the Institutional Review Board of People’s Hospital of Zhengzhou University.

5 mL blood samples were collected from each subject for genomic DNA extraction using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions, and stored at −20 °C. Target region capture and high-throughput sequencing were examined in the proband (III:6) for 131 previously reported genes associated with neuromuscular disease (Figs. 2 and 3). Briefly, point mutations and insertions/deletions were designed for target genes encompassing all exons and splicing sites immediately adjacent to intron sequences. Screening for mutations in the target genes was performed by next generation sequencing (NGS) (Illumina HiSeq 2500, USA) with ≥ 200-fold average high-throughput sequencing depth; mutations were analyzed by bioinformatics.

Sanger sequencing was used to detect DNA sequence variants; then, the target site primer was designed based on the location of the SYNE1 gene variant. 50 μL aliquots of standard polymerase chain reaction (PCR) reactions contained 25 μL Premix Taq, 2 μL each forward and reverse primers, 5 μL genomic DNA as template, and 16 μL ddH₂O. To assess PCR efficiency, 8 μL of amplified products were analyzed by 2% agarose gel electrophoresis. PCR primers were: forward 5′-GGAGCTGGCTTCCATGATGAG-3′ and reverse 5′-ACGTGTTGCTTACTTTGCTGT-3′. PCR amplification was performed on MultiGene OptiMax (Labnet, USA) at 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 1 min. Subsequently, the PCR products of the proband and five family members were purified and directly sequenced on an automated sequencer. Sequencing reads were compared with the Chromas software and NCBI BLAST.

The proband (III: 6) was a 24-year-old male. From 12 years of age, he had progressive and bilateral weakness of proximal lower limbs, and proximal muscles of the extremities gradually degenerated. Gradually, he experienced fatigue after walking a short distance, which was slowly accompanied by waddling gait. Subsequently, the neck could not move forward, and the elbow could not be kept straight. Contractures of the bilateral ankle, knees, and elbows appeared. Then, he started to pull himself up by gripping handrails while ascending the stairs, at the age of 16 years. He had difficulties standing up after squatting on the ground. The symptoms mentioned above worsened progressively. At neurological examination, the patient had normal intelligence. The neck could not move forward, and head activities were limited. The bilateral ankle, knee and elbow showed contracture. The hip, wrist, and shoulder were roughly normal. The biceps brachii and quadriceps femoris had significantly shrunken. Muscle tension of both upper limbs was normal, and that of both lower limbs was reduced. The proximal muscle strength of both upper limbs was grade 4; the distal muscle strength was grade 5. The bilateral biceps, triceps, and tendon reflexes had disappeared, and radial periosteal reflex on both sides could be derived. Bilateral knee and ankle reflexes had also disappeared. Relevant laboratory results showed serum muscular enzyme levels distinctly increased (3252.0 u/L), elevated by 17-fold. Blood lactic acid levels were increased slightly (3.5 mmol/L). Magnetic resonance imaging examination of muscles showed bilateral thigh muscle disappearance; muscles were filled with large amounts of adipose tissue while the anteromedial calf group of muscles were infiltrated mildly with fat (Fig. 4 A1-4). Electromyography revealed normal nerve conduction and typical muscle-derived changes, such as low amplitude, broad phase, and significantly increased polyphasic wave. Electron microscopy demonstrated that the size of muscle fibers (obtained by biceps brachii biopsy) was not uniform, and fiber atrophy and hypertrophy existed alternately. Compensatory hypertrophy of partial muscle fibers was found, with apparent fat and connective tissue proliferations. Split muscle fibers could be seen in the hypertrophy muscle fibers (Fig. 4 B1-2). Cardiac examinations were normal (Fig. 5). Patient 2 (III: 5) was a 28-year-old female with proximal weakness of bilateral lower limbs. Neurological examination showed muscle wasting. The clinical cardiac evaluation revealed no abnormalities (Fig. 5). Patient 3 (II: 3) was a 53-year-old female with proximal weakness of bilateral lower limbs, and would feel fatigued after slowly walking a short distance. Clinical cardiac evaluation showed no abnormalities (Fig. 5).

Target region capture sequencing showed a heterozygous missense mutation in exon 46 of the SYNE1 gene (NM_033071, c.6910A > G, p.G2304R) of the proband, which lead to a glycine-to-arginine substitution (p.Gly2304Arg) that has been associated with the EDMD-like phenotype. The mutation were present in two other members of the family (II3 and III5) (Fig. 6c) but not in the other 3 family members or the 100 Chinese individuals as healthy controls. The mutation was not detected in the remaining 3 pedigrees or the 100 healthy individuals. This missense mutation was not reported in The Human Genetics Mutation Database (http://​www.​hgmd.​org; [8]) and previous literatures. In addition, the mutation was not a single nucleotide polymorphism. Furthermore, mutation analysis by the Polyphen-2 online software and MutationTaster, HumDiv (sensitivity: 0.86; specificity: 0.91) and HumVar (sensitivity: 0.88; specificity: 0.75) predicted potentially damaging effects, which indicated that the missense mutation might be pathogenic. The SYNE1 protein sequences of six mammalian species, including human, were examined and analyzed by NCBI GenBank (Fig. 6a). The glycine at position 2304 of the SYNE1 protein was located in a highly conserved region. The more mutations in the conserved region, the more pathogenic the gene. Therefore, we hypothesized that G2304R may be a pathogenic mutation. This mutation altered the side chain of the 2304 amino acid residue as assessed by the Raptor 3D online software (Fig. 6b). These results demonstrated that the novel mutation was the most probable cause of the familial EDMD-like.