Four consecutive patients of SEPN1-RM were collected from April 2019 to May 2021 from the Xuanwu Hospital. Clinical evaluations and neurological examinations were performed by the two senior neurologists. This study was approved by the Ethics Committee of Xuanwu Hospital, Capital Medical University. Written informed consents were obtained from all the participants. Detailed medical history, routine laboratory tests, electromyogram (EMG), and MRI of the muscle were collected.

Muscle biopsies from the biceps brachii or quadriceps femoris were obtained from four cases. Frozen sections of the mass specimen were processed according to the standard protocols and stained with hematoxylin-eosin staining (HE), Gomori Trichrome, nicotinamide adenine diphosphate hydride (NADH), succinate dehydrogenase (SDH), cytochrome oxidase (COX), periodic acid-Schiff (PAS), and ATPase for light microscopic investigations (2). Further immunohistochemical stainings with dystrophin R, C, N and desmin were processed.

Peripheral blood samples were collected from the patients and other family members. Genomic DNA was extracted from ethylenediamine tetraacetic acid (EDTA) blood. Mutation analysis was performed by whole-exome sequencing and confirmed by Sanger sequencing (11). Compound heterozygous mutations of SELENON gene were identified in all the patients and confirmed by familial segregation analysis. Variants were reported by using the complete SEPN1/SELENON transcript (NM_020451.2). Novel mutations were evaluated by the variant-classification guidelines of the American College of Medical Genetics and Genomics (ACMG) guidelines (12). The Genome Aggregation (http://gnomad.broadinstitute.org/) and the Clinvar (http://www.ncbi.nlm.nih.gov/clinvar/) databases were interrogated to identify the previously reported mutations and to determine variant frequency in the population. Pathogenicity of the variants was determined based on population frequency and in silico prediction programs (Protein Variation Effect Analyzer (PROVEAN), http://provean.jcvi.org/index.php; Polyphen-2c, http://genetics.bwh.harvard.edu/pph2/; Mutation Taster, http://www.mutationtaster.org; 1,000 Genomes, http://www.1000genomes.org; ExAC Browser, http://exac.broadinstitute.org; The Human Gene Mutation Database (HGMD), http://www.biobase-international.com/product/ hgmd; and SpliceAI, https://github.com/Illumina/SpliceAI).

For a better delineation of heterogeneous phenotype and extramuscular abnormalities in patients with SEPN1-RM, literature review was performed in PubMed to find related articles from first available to August 20, 2021. Search terms included “SELENON” or “SEPN1” or “SEPN1-RM” or “Selenoprotein N-related myopathy” in all the fields. The full list of reported cases and corresponding variants was listed in Supplementary Table 1. Only cases described or mentioned delayed respiratory dysfunction or extramuscular abnormalities were abstracted in literature review part of this study. For each case related, we documented the basic information, clinical descriptions, and genetic data, if information was available. We excluded: (1) publications without English abstract, (2) reviews or fundamental studies with no clinical information, (3) clinical studies provide insufficient data, and (4) cases without genetic confirmed diagnosis. If one patient was reported in different studies, only the study providing more sufficient information was included. Only data that were explicitly stated in studies were abstracted.

A total of four male patients included in this study were aged 14–36 years including two juvenile cases (patients one and four) and two adult cases (patients two and three). Each of them was born from non-consanguineous patients with unremarkable family history. Their detailed clinical data are listed in Table 1. Age of onset ranged from birth to the first decade with initial symptom to be abnormal gait in patient one, delayed motor milestones in patient four, and poor sports performance in patients two and three. Symptoms of early myopathy were underrecognized, especially in patients two and three who led a normal life until the 3rd decade. All the patients presented with mild fatigue, axial and limb girdle weakness, scoliosis, rigid spine, and respiratory insufficiency. Clinical investigations disclosed reduced tendon reflexes, positive Gowers' sign, and waddling gait. As for auxiliary examination, normal or mild elevated serum creatine kinase (CK) levels (166–634 IU/L), reduced Forced Vital Capacity (FVC) (21.3–60%), and myopathic alterations in EMG were revealed. At 6 months follow-up, except for patient one who was still free of Non-invasive ventilation (NIV), other patients were supported with intermittent NIV and achieved improvement in daily activities.

Clinical severity and disease progression vary between individuals. The two adolescent patients exhibited early myopathy and puberty aggravation. Patient one presented abnormal gait and suffered frequent falls at 5 years old. Scoliosis developed at the age of 12 years (Figure 1A) and slight night apnea appeared at the age of 14 years. Spinal rigidity was observed in cervical segment with limited mobility in head rotation. Patient four developed nocturnal hypoventilation, episodes of pulmonary infections, and hypoxemic and hypercapnic respiratory failure at the age of 14 years. During hospitalization, he experienced hypercapnic coma and relieved after assisted ventilation. Mild scoliosis and spinal rigidity were found. The two adult patients showed a chronic disease progression. Patient two felt normal until the age of 34 years when noticing difficulty in raising up after bending over. Later, he developed spine rigidity (Figure 1B), proximal lower limbs weakness, and progressively nocturnal dyspnea. He was diagnosed and initiated NIV at the age of 37 years. Patient three was a 29-year-old male with lower limbs weakness from the age of 22 years. He exhibited progressively nocturnal dyspnea and started NIV at the age of 29 years. No prominent scoliosis and spine rigidity were found.

Extramuscular abnormalities were observed and raised challenge for diagnosis in patients one to three. Typical features of low body weight and overall amyotrophy in SEPN1-RM were not observed in our patients. On the contrary, weight gain and fat tissue accumulation were revealed in three patients. Patient one had good appetite and gained much weight during puberty. He was a short stature with a BMI of 23.15 kg/m² (height 152 cm and weight 53.5 kg) (13). Subcutaneous adipose tissue was accumulated in his chest. Patients two and three both experienced a rapid weight gain before symptoms worsened with increased dietary intake and reduced daily activities (patient two: from 53 to 73 kg at the age of 34 years; patient 3: from 50 to 65 kg at the age of 21 years). They both showed prosperously swollen abdomen (BMI of 25.3–25.4 kg/m², overweight). Auxiliary examination ruled out ascites, cardiac dysfunction, thyroid problems, and diabetes mellitus. The second extramuscular symptom was mental disability. Upon entering primary school, patient one showed bad learning performances. Simple addition and subtraction within 10 were incapable. Recognition impairment was confirmed by low scores of the Wechsler intelligence question (IQ) test (IQ = 45). MRI of the brain and metabolic screening revealed no significant findings. In addition, mild cardiac abnormalities were revealed in patient three. Cardiac ultrasonography showed slightly enlarged right atrium (42 mm) and severe pulmonary arterial hypertension (72 mm Hg). Holter monitoring revealed sinus bradycardia, nodal tachycardia, and intermittent second-degree atrioventricular block (Supplementary Figure 1).

Magnetic resonance imaging of the muscle was performed in patients one to three and revealed similar involvement pattern with various severities (Figures 1C–I). Muscle mass was overall reduced and subcutaneous adipose was accumulated. Bilateral gluteus maximus (Figures 1D–F) and sartorius (Figures 1H,I) were the most commonly affected, while adductor longus and rectus femoris were often preserved even in severe lesions. The posterior compartment tends to be more seriously involved. The MRI profile was compatible of previous findings and showed no correlation with clinical severity (14–16). All the patients underwent muscle biopsy and pathological examination. The most prominent feature was the multiminicore lesions in NADH and SDH staining, which was observed in all the patients. Core changes were mostly distributed in atrophy fibers with different sizes. Other unspecific morphological changes included fiber size variations, mild increased connective tissues, type 1 fiber hypotrophy, eosinophilic inclusions immunoactive for desmin, and occasional blue fibers. Immunohistochemical staining of dystrophin was normal in all the patients. The pathological features are illustrated in Figures 2A–F.

In all patients, compound heterozygous mutations of SELENON gene were identified by whole-exome sequencing, which were inherited from their parents, respectively. Three novel mutations and their pathogenic classification are listed in Table 2. All of them were absent from the control database. Patient one carried a reported pathogenic mutation c.1574 T>G (17) and a novel in-frame mutation c.1286_1288 del CCT. The latter induced a deletion of serine in 429 residue in a non-repeat region, expecting to produce a protein missing a single amino acid (p.S429del). The deleted serine was located upstream the UGA codon and not conserved itself. However, it is included within a block of strictly conserved residues, likely inducing a direct effect on Selenoprotein N structure and activity. Patient two identified a previously reported critical selenocysteine mutation c.1384T>C (p.U462R) (18) and a novel duplication c.1078_1086dupGGCTACATA (p.I362_P363insGYI). His sister was a healthy carrier of the novel mutation. It caused an in-frame insertion of the glycine, tyrosine, and isoleucine, likely producing a longer protein of 593 amino acids. It was predicted to have deleterious effects. Patient three carried heterozygous mutations of c.943 G>A and c.785 G>C. The first has been reported pathogenic (19). The latter was a novel missense mutation inducing a substitution of the highly evolutionary conserved arginine to a proline at codon 262 (p.R262P). This amino acid change constitutes a major physicochemical modification and was predicted to be disruptive on protein structure and function. Patient four identified c.1406G>A(p.R469Q) and c.802 C>T(p.R268C) of SELENON gene, both were reported in SEPN1-RM (5, 20). His healthy sister has not been tested. All the three novel mutations had probably no impact on splicing based on SpliceAI prediction (SELENON chr1: 26139181-26139184 TCCT>T SpliceAI = T|SELENON|0.00|0.00|0.00|0.00|39|-3|-16|-4, chr1: 26138020 dup A AGGCTACATA SpliceAI = AGGCTACATA|SEPN1|0.00|0.00|0.00|0.00|0|7|33|6, chr1: 26135554 G>C SpliceAI = C|SEPN1|0.05|0.01|0.00|0.00 |14|-37|-38|-16).

In the literature review, we found 10 cases of SEPN1-RM presenting delayed respiratory insufficiency (Table 3) (2–4, 10, 21–23). These patients were globally distributed and ranged from 26 to 71 years old. Nocturnal dyspnea and NIV assistance were postponed to the third to eighth decades of life, sometimes after predisposing events such as stroke, acute respiratory failure, and pregnancy. All these patients presented relatively mild symptoms of an infantile myopathy including muscle weakness, spinal rigidity, scoliosis, and contractures. No correlation was found between the mutation type and clinical severity.

There were 18 studies mentioned extramuscular abnormalities of SEPN1-RM in current literature (Table 4). Since this aspect has rarely been focused, literature review only provided a brief overview rather than quantitative analysis. Overall, joint contractures/hyperlaxity and body composition abnormalities seem to be the most common extramuscular symptoms (4). In patients with altered body composition, low BMI/body weight was frequently reported, while overweight cases or steep weight gain were quite rare (4, 22). Some patients showed short stature and altered fatty tissue distribution (6, 14). Additional metabolic changes included insulin resistance and osteoporosis (6, 7, 28). A few patients presented cardiac abnormalities of which mostly were mild or secondary to respiratory failure (4, 10, 22, 28). Three cases combined intellectual disability or developmental delay (8, 18).