A novel homozygous exon2 deletion of TRIM32 gene in a Chinese patient with sarcotubular myopathy: A case report and literature review

Sarcotubular myopathy (STM) is a rare autosomal recessive myopathy caused by TRIM32 gene mutations. It is predominantly characterized by the weakness of the proximal limb and mild to moderate elevation of creatine kinase levels. In this study, we describe a 50-year-old Chinese man who exhibited a proximal-to-distal weakness in the muscles of the lower limbs and who had difficulty standing up from a squat position. The symptoms gradually became more severe. He denied a history of cognitive or cardiological problems. The patient’s parents and children were healthy. Histopathological examination revealed dystrophic changes and irregular slit-shaped vacuoles containing amorphous materials. Whole-exome sequencing consisting of protein-encoding regions of 19,396 genes was performed, the results of which identified one novel homozygous 2kb deletion chr9.hg19: g.119460021_119461983del (exon2) in the TRIM32 gene. This was confirmed at the homozygous state with quantitative real-time polymerase chain reaction. Here, we present a Chinese case of STM with one novel mutation in TRIM32 and provide a brief summary of all known pathogenic mutations in TRIM32.

INTRODUCTION Sarcotubular myopathy (STM; MIM 268950) is a rare autosomal recessive disease characterized predominantly by weakness of the proximal limb. Mutations in the TRIM32 gene, which encodes a protein consisting of 653 amino acids residues, are often associated with STM and the limb-girdle muscular dystrophy R8 (LGMDR8, previously known as LGMD2H; MIM 254110) [1], two conditions that present as different severities of the same disease [2]; Bardet Biedl syndrome presents a different phenotype due to one variant (P130S) in the B-box region, independently from muscular phenotypes [3]. Since the first case was reported in Jerusalem in 1973, STM has only been described in 28 patients across the world [2,[4][5][6][7][8][9][10].
TRIM32 is a member of the tripartite motif (TRIM) family and is composed two exons, with the entire open reading frame located within exon2. The TRIM32 protein sequence contains several conserved N-terminal motifs including a RING-finger, a B-box, and a coiled-coil region, followed by six NHL repeats at the C-terminus [11]. Various mutations have been reported to cause TRIM32-related myopathy, and the majority of these patients are of Hutterite origin and overwhelmingly homozygous for the c.1459G >A (p. D487N) mutation [1,12]. However, additional mutations that cause LGMDR8/STM have since been documented in different populations [6,[8][9][10][13][14][15]. To date, no disease-causing mutations in the TRIM32 gene have been reported in the Asian population. A total of 27 different mutations have been reported in patients with TRIM32related myopathy, including one nonsense, ten missense, nine frameshifts, two small deletion, and five large deletion variants (Table S1). Apart from the most frequent D487N mutation and all the recently identified single nucleotide pathogenic variants in the C-terminal NHL domain, a relatively high frequency of frameshift mutations have been reported outside the NHL repeats.
Herein, we identified a Chinese patient with a new homozygous deletion mutation in exon2 and described the clinical, pathological, and image-based findings of his clinical assessment. This is the first independent report of TRIM32-mutated STM in China.

CASE REPORT
A 50-year-old male was admitted to our hospital complaining of slowly progressing weakness. He stated that when www.bjbms.org limb muscles demonstrate minimal fatty infiltration and no distinct involvement pattern ( Figure S1C-D).
Peripheral blood samples were obtained with informed consent from three members of the family. Genomic DNA was isolated from these samples according to standard techniques. All known protein-encoding regions, including a total of 19,396 genes, were captured and enriched. The enriched libraries were sequenced on an Illumina NovaSeq 6000 (Illumina, USA), which generated data covering 99.9% of the sequence at 300×. The sequencing reads were aligned on a reference human genome (hg19) using Burrows-Wheeler Aligner software. The Genome Analysis Toolkit (GATK) was used to detect single-nucleotide variants and indels of the reads. Whole exome sequencing was performed at Chigene Medical Research Centre (Beijing, China). It turned out that the patient had homozygous deletion of a 2kb region, chr9. hg19: g.119460021_119461983del, in exon 2 of the TRIM32 gene ( Figure 1A). The presence of this homozygous deletion was confirmed with quantitative real-time polymerase chain reaction ( Figure 1B). The patient' s parents were heterozygous for the mutation. This novel mutation was neither observed in 200 healthy Chinese controls, nor four public databases, including dbVar, ClinVar, the Database of Genomic Variants (http://dgv.tcag.ca/dgv/app/home), and the Leiden Open Variation Database (www.lovd.nl/TRIM32).
After written informed consent was obtained from the patient, a gastrocnemius biopsy was conducted. Muscle he was 4 years old, his mother noticed that he was not to be able to run as fast as his peers. He was the first of three children of his non-consanguineous parents, and both his siblings and his parents were healthy. No other muscular symptoms were noted throughout his childhood. In the 2 years leading up to his evaluation, he noticed mild weakness in his legs and difficulty getting up from a squatting position. These symptoms gradually became more severe, until he presented at age 50 with proximal-to-distal weakness in the muscles of the lower limbs. He has never experienced cognitive or cardiologic problems.
Neurological examination revealed mild proximal muscle weakness in the lower extremities and the toe extensor muscles (MRC grade 4). The Gower' s sign test was positive and his tendon reflexes remained present but weak. There was no evidence of calf hypertrophy, scapular winging, or facial, and ocular impairment. Laboratory testing revealed slightly elevated creatine kinase levels (2× normal). Electromyography abnormalities of myopathic changes were found in both the vastus medialis and tibialis muscles. Echocardiograph and electrocardiogram results were normal. A magnetic resonance imaging (MRI) of the patient' s muscles revealed a preferential fatty infiltration in the posterior compartment of the patient' s thigh, with marked sparing in the gracilis, sartorius, and quadriceps muscles ( Figure S1A). There was more focal involvement in the gastrocnemius muscles, and the remaining muscle groups were mildly involved ( Figure S1B). The upper FIGURE 1. TRIM32 deletion mapping and mRNA expression. Whole exome sequence data showing the homozygous deletion of the coding region of exon 2 in the TRIM32 gene, which leads to a total absence of amplification and a ratio of zero for the probe covering this region (A). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of TRIM32 messenger RNA expression in blood. Messenger RNA levels were quantified with real-time PCR and normalized to the levels of ALB. A significant reduction in real-time PCR quantification of TRIM32 gene expression is observed in the patient's blood compared with unaffected controls (B). B A www.bjbms.org biopsy revealed marked variation of the muscle fiber diameters, internalized nuclei, as well as necrotic and atrophic myofibers. Small and irregularly slit-shaped vacuoles containing basophilic material were observed throughout the sarcoplasm in scattered myofibers. The connective tissue of the muscle was moderately increased. However, no inflammatory cell infiltration was observed (Figure 2). Immunohistochemical staining showed that anti-dystrophin-N, -C, -R proteins, anti-α-, β-, γ-, δ-sarcoglycan proteins, calpain-3, and dysferlin levels were normal.

DISCUSSION
Our patient' s STM diagnosis is based on limb-girdle weakness, the presence of vacuoles in his skeletal muscle biopsy, and genetic testing. To our the best of knowledge, this is the first report outlining the diagnostic characteristics of STM in a patient of Chinese ethnicity, and the 29 th report worldwide that assesses STM.
The number of reports, including systematic literature reviews, on pathogenic TRIM32 mutations is limited. Since TRIM32 mutation-related disorders were first described by Frosk et al. in 2002, 81 total cases have been formally reported. TRIM32 gene mutations and their associated phenotypes are summarized in Figure 3 and Table S1. TRIM32 mutations are most commonly associated with LGMDR8 (68% [55/81] of the cases), followed by STM (30% [24/81] of the cases). Furthermore, STM generally has an earlier onset characterized by more severe weakness than LGMDR8 [12,15,16]. As previously reported by Nectoux et al., although the proband became wheelchair-bound in the late course of his disease, a relatively mild phenotype, as in the current patient, has also been noted [17]. Our patient was still able to walk without support, suggesting that the clinical manifestation of individuals with homozygous deletions of TRIM32 may not correlate with a higher severity in phenotype. Therefore, the relationship between the type of mutation and the resulting phenotype should be investigated further.
Nectoux et al. reported a patient with early-onset LGMDR8 and mild cognitive deterioration who had a homozygous 336 Kb deletion that encompassed the entire TRIM32 gene and part of the ASTN2 gene [17]. They postulated that the cognitive disability could be related to a complete loss of function of TRIM32/ASTN2 activity. Conversely, our patient did not have any evidence of cognitive impairment, including no abnormal difficulty with concentration or weakness in immediate memory for his age. This discrepancy in phenotypes suggests that cognitive impairment should be associated with ASTN2 loss of function rather than TRIM32 loss of function. However, further investigation is needed to verify this novel finding. Together with the previously published report, our results indicate that a homozygous deletion variant is associated with pathogenicity of TRIM32-related myopathy.
In summary, we report a novel TRIM32 mutation in a Chinese patient with STM and describe the differences in clinical phenotype between a homozygous deletion mutation and other previously identified mutations. This observation  suggests that TRIM32 inactivation may not influence cognitive impairment in STM patients. We also provided MRI evidence of the upper limb muscles from a TRIM32 mutated patient. This finding broadens the mutational and phenotypic spectra of TRIM32-related myopathies and can guide proper genetic counseling for pre-and post-natal screenings in the future.