A novel EMD mutation in a Chinese family with initial diagnosis of conduction cardiomyopathy

Abstract Introduction Emery–Dreifuss muscular dystrophy (EDMD) is a hereditary myopathy characterized as triad of muscular dystrophy, joint contractures, and conduction cardiomyopathy. In this study, we diagnosed a X‐linked recessive EDMD patient with severe conduction cardiomyopathy while noteless muscular and joint disorders. Methods A Chinese cardiomyopathy family spanning four generations was enrolled in the study. Targeted next‐generation sequencing (NGS) was performed to identify the underlying mutation in the proband and validated by Sanger sequencing. Segregation analysis was applied to all 13 participants. Results A novel frameshift mutation (c.253_254insT, p.Y85Lfs*8) of emerin gene (EMD) was found and co‐segregated with family members. Other than the typical manifestations of X‐linked EDMD, this patient presented inconspicuous muscular disorders which were later diagnosed after the mutation been identified. Conclusions This study enriches the EMD gene mutation database and reminds us of the possibility of EDMD while encountering patients with severe heart rhythm defects or dilated cardiomyopathy of unknown etiology, even if they have neither obvious skeletal muscle disorder nor joint involvement.

The potential mutations from NGS were validated by Sanger sequencing. Segregation analysis was applied to all participants. Primer pairs were designed by Primer 3, PCR amplifications were performed with forward primer [5'-GGGGCAAACAGTTCTGTCTC-3'] and reverse primer [5'-AGAGCCACCATTTGTACCCA-3']. PCR products were checked on agarose gels and sequenced on an ABI 3,730 DNA sequencer (Applied Biosystems, Foster City, CA, USA). The sequencing results were compared with gene reference sequences in the UCSC hg19 to confirm potential mutations. NM_000117.2 was used as the reference sequence for the coding regions of the EMD gene.

| Clinical findings
The proband (III.3 in Figure 1), a 50-year-old man, who suffered from occasional palpitations, lower extremities edema and anhelation for

| Genetic findings
Mutation analysis identified an 1-bp insertion (c.253_254insT) in EMD gene exon 3, the frameshift hemizygous mutation resulting in a premature stop codon (p.Y85Lfs*8). Familial aggregation analyses revealed the mutation presented in 5 family members, including two hemizygotes (III.3, III.14) and three female carriers (II.2, II.12, IV.2). The propagating analysis revealed the mother carried the EMD heterozygous mutation. Subsequently, the patient's symptomatic cousin took examinations and found elevated CK level (458.9 U/L, reference range 50-310 U/L) and roughly normal electrocardiography (ECG). All the female carriers and his unaffected son had normal F I G U R E 1 Pedigree of the family and mutation in EMD gene. Sanger sequence revealed a 1-base insertion at c.253 in exon 3 of EMD ECG and CK levels. The results verified the genotypes co-segregated with phenotypes.
Half a year later, the proband received muscle biopsy on the left deltoideus triangularis. Pathological diagnosis showed myogenic damage, which was conformed to myogenic fibrous myopathy

| D ISCUSS I ON
X-linked EDMD is mainly caused by EMD mutations. The EMD gene (NM_000117.2) is mapped to chromosome Xq28 and contains 6 exons. The gene EMD encodes a ubiquitous nuclear membrane protein emerin consisting of 254 amino acids, which participates in membrane anchorage to the cytoskeleton (Bione et al., 1994). It was reported that cardiac involvement is the most important clinical symptom among patients with EMD mutations (Ura et al., 2007;Yuan et al., 2014). Cardiomyopathy appears in early adulthood and usually manifests as heart conduction block. Cardiac involvement usually arises after the second decade of life (Ben Yaou et al., 2007). ECG abnormalities occurrence often precede muscle disease. Cardiac conduction block can occur as early as 12 years of age and its severity is not related to the amount of muscular weakness and contractures (Sumitani, Ishikawa, Ishikawa, & Minami, 1995). Lack of emerin levels in heart may alter electrophysiology and myocardial cell adhesion, which could lead to conduction block (Sakata et al., 2005).
X-linked EDMD associated with EMD mutations is clinically heterogeneous, ranging from mild to severe forms (Meinke et al., 2015).
Different from the typical clinical features, the proband in our study had a medical history of severe arrhythmia and dilated myopathy, while inconspicuous skeletal muscular weakness and normal serum creatine kinase level. The other affected male (III.14), who presented paroxysmal cardiopalmus and slight limb weakness, had currently normal ECG, should be under close monitoring of cardiac function.
Prognosis in EDMD is strongly associated with the severity of cardiac involvement. Continuous follow-up of cardiac function is essential, including female carriers even in the absence of distinct clinical signs. It is crucial to provide prompt cardiac intervention and avoid unexpected lethal heart diseases for these particular patients. This There are only few case reports on the predominant cardiac features of EDMD patients with EMD mutations (Karst, Herron, & Olson, 2008;Sakata et al., 2005;Vohanka et al., 2001;Yuan et al., 2014;Zhang et al., 2014) (Table 1). Learning from the previous reports, we knew that EMD mutations might be responsible for independent cardiac phenotype with severe heart rhythm defects or presenting as dilated cardiomyopathy, while muscle involvement and joint contractures were mild, localized, or seldom noticeable until the mutation been identified. It is interesting to find that four of the six reported patients were from Asia, whether the atypical X-linked EDMD manifestations were due to incomplete penetrance or racial differences are still unknown. Our findings increase the case numbers of these rare clinical features and may provide research value of Asian patients in EDMD. Although EMD gene is specifically associated with X-linked EDMD, the genotype-phenotype correlations, with special reference to cardiac involvements, still need to be further investigated.
In conclusion, we describe a novel mutation of EMD gene, which provides genetic diagnosis for the atypical X-linked EDMD individuals of the family. We provided scientific guidance and promote early intervention for EDMD patients, which may minimize the harmful effects of the disease.

ACK N OWLED G M ENTS
We thank all subjects for participating in this study. We thank Ping Xue from the Department of Surgery Research, the Second Hospital of Hebei Medical University for her assistance of muscle histopathology.

CO N FLI C T O F I NTE R E S T
None Declared.