Novel SPEG variant cause centronuclear myopathy in China

Abstract Background Centronuclear myopathy (CNM), a subtype of congenital myopathy (CM), is a group of clinical and genetically heterogeneous muscle disorders. Centronuclear myopathy is a kind of disease difficult to diagnose due to its genetic diversity. Since the discovery of the SPEG gene and disease‐causing variants, only a few additional patients have been reported. Methods A radiograph test, ultrasonic test, and biochemical tests were applied to clinical diagnosis of CNM. We performed trio medical exome sequencing of the family and conservation analysis to identify variants. Results We report a pair of severe CNM twins with the same novel homozygous SPEG variant c. 8710A>G (p.Thr2904Ala) identified by clinical trio medical exome sequencing of the family and conservation analysis. The twins showed clinical symptoms of facial weakness, hypotonia, arthrogryposis, strephenopodia, patent ductus arteriosus, and pulmonary arterial hypertension. Conclusions Our report expands the clinical and molecular repertoire of CNM and enriches the variant spectrum of the SPEG gene in the Chinese population and helps us further understand the pathogenesis of CNM.


| INTRODUC TI ON
Centronuclear myopathy (CNM) is a subgroup of congenital myopathy, 1 characterized by skeletal muscle weakness and atrophy. 2 The phenotypes of CNM are commonly present at newborn, infant, and early childhood.
SPEG is a protein highly expressed in striated muscle cells and cardiomyocytes, which play an important role in the differentiation of vascular smooth muscle cell in early life. [9][10][11][12] Recessive SPEG (Striated muscle enriched protein kinase) mutations were first identified in CNM patients by Agrawal in 2014. 13 To date, SPEG mutations have been identified in eight CNM and 1 CM patients. [13][14][15][16][17] Most of these patients had severe hypotonia and muscle weakness.
Here, we report an additional pair of fraternal CNM twins with a novel homozygous SPEG variant, c.8710A>G (p.Thr2904Ala). We have also reviewed clinical findings of reported 11 CNM patients and discussed the genotype-phenotype correlations, to further our understanding of SPEG-related CM.

| Specimens and DNA preparation
Blood samples were collected and prepared at Jiangmen Maternity and Child Health Care Hospital. Approval of the Jiangmen Maternity and Child Health Care Hospital ethics committee was obtained, as well as informed consent from all adult participants and parents of the participating children. Genomic DNA was extracted from peripheral blood using the Solpure Blood DNA kit (Magen) according to the manufacturer's instructions.

| Variant annotation and interpretation
The raw reads were filtered to generate "clean reads" by removing adapters and low-quality reads (Q20). Clean reads were mapped to the reference human genome hg19 (2009-02 release) with BWA 0.7.15. Unmapped reads were removed. SNVs and short indels were scored and reviewed by using NextGENe 2.4.1.2 and GATK 3.5. In addition, the eCNVscan was used to detect large exonic deletions and duplications. 19 The normalized coverage depth of each exon of a test sample was compared with the mean coverage of the same exon in the reference file, to detect copy number variants (CNVs). 20 All short variants were annotated with databases including 1000 Genomes, dbSNP (build 148), gnomAD (http://gnomad.broad insti tute.org/), ClinVar, HGMD, and OMIM.

RT-PCR
Genomic DNA was extracted from whole-blood samples.
Mutagenesis was carried out according to the PCR mutagenesis protocol Site-directed mutagenesis. Wild-type (wt) minigene SPEG exon 36 with intronic was used as template to generate variants. We subjected 36 exons of SPEG with the intronic boundaries to PCR-Sanger sequencing. PCR was performed under the following conditions: initial denaturation at 95°C for 3 minutes, followed by 35 cycles of 95°C for 30 seconds, 62°C for 30 seconds, and 72°C for 30 seconds.  Table S1. Each PCR product was digested with the BglII and MluI restriction enzymes and cloned into the pCAS2 vector, which had also been digested with BamHI and MluI. All of the selected clones were sequenced, and the verified clones, referred to as the wild-type (pSPEG-c.8710A) and mutant (pSPEG-c.8710G) clones, were retained for expression experiments.
Then, 1.5μg of total RNA was reverse transcribed using a Reverse Transcription System according to the manufacturer's instructions (Invitrogen). Following RNA retrotranscription, 200 ng of complementary DNA from the three constructs mentioned above was PCR amplified using the primers (reverse transcriptase-PCR-F/R) shown in Table S1. The PCR products were then separated on a 1% agarose gel, and individual bands were excised and sequenced using specific primers (SEQ primer-F/R, Table S1). 22,23

| Clinical description
Parents of our twin patients have three pregnancies. The previous two pregnancies resulted in abortion. The third pregnancy resulted in the fraternal twins described here. ( Figure 1A). During the first pregnancy in 2011, the fetus was found to have strephenopodia and limb deformities by ultrasound at 24th week, and abortion was performed.
During the second pregnancy in 2013, mother reported decreased fetal movement. Ultrasound showed limb deformities and strephenopodia at 25th week of pregnancy, and abortion was performed.
Karyotype analysis of the two fetuses showed no chromosomal abnormalities.
During the third pregnancy in 2016, mother reported fetal akinesia. Ultrasound showed twin with strephenopodia and increased echogenicity of the subcutaneouslayer and muscularlayer

| Genetic results
Genetic counselling was provided, and informed consent was ob-

| Impact of the c.8710A>G mutation on SPEG mRNA splicing
Mutation taster result of c.8710A>G show may change splice site and effect SPGE mRNA splicing. An in vitro minigene system was used to variant c.8710A>G effect on splicing, and the experiment result showed no effect on splicing.  Table 1.

| D ISCUSS I ON
The interaction with MTM1 was considered the main pathogenic pathway of SPEG function. The region in the C-terminal (amino acid 2530-2674) is required for interaction with MTM1. These can explain most genotype-phenotype correlations of the patients. It was discovered in a mouse that SPEG dysfunction produces a myopathy by affecting Ca 2+ current function of the voltage sensor, calcium release from the SR and consequently reducing muscle contractility. 24,25 Recent studies confirmed that the protein kinase domain II is actually the key domain that controls the Ca 2+ re-uptake through regulating SERCA2a. 26 These indicate that dysfunction of protein kinase domain II of SPEG may cause CNM because of unbalanced calcium homeostasis through the SERCA2a pathway. The mutation p.Arg3196*, p.Val3062del, p.Val2997Glyfs*52 also occurred just before the interaction region with MTM1 and may cause dysfunction of protein kinase domain II.
SPEG is alternatively spliced into four tissue-specific isoforms that were identified in murine models including SPEGα, aortic preferentially expressed gene-1 (APEG-1), SPEGβ, and brain preferentially expressed gene (BPEG). SPEG has a critical role in skeletal and cardiac function, and SPEGα and SPEGβ are highly expressed in skeletal and cardiac muscle. 10 Clinical data from Patients 4, 8, and 10 suggest that SPEGα may partially rescue mutations affecting only SPEGβ, possibly preserving cardiac function. SPEGα and SPEGβ are proteins within the junctional membrane complex (JMCs) that reported to regulatory junctophilin-2 (JPH2) phosphorylation, which is a key role for transverse tubules maintenance in cardiac myocytes. 16 Clinical data from patients 3,5-7,9,11 carried mutation affecting SPEGα and SPEGβ. These findings suggest the disease is more severe when both isoforms are affected. Our two patients who carried variant p.Thr2904Ala affecting SPEGα and SPEGβ were severe and died within a week.
In conclusion, we diagnosed a CNM family and pathogene-