Case report: A novel patient presenting TRIM32-related limb-girdle muscular dystrophy

Limb-girdle muscular dystrophy autosomal recessive 8 (LGMDR8) is a rare clinical manifestation caused by the presence of biallelic variants in the TRIM32 gene. We present the clinical, molecular, histopathological, and muscle magnetic resonance findings of a novel 63-years-old LGMDR8 patient of Italian origins, who went undiagnosed for 24 years. Clinical exome sequencing identified two TRIM32 missense variants, c.1181G > A p.(Arg394His) and c.1781G > A p.(Ser594Asp), located in the NHL1 and NHL4 structural domains, respectively, of the TRIM32 protein. We conducted a literature review of the clinical and instrumental data associated to the so far known 26 TRIM32 variants, carried biallelically by 53 LGMDR8 patients reported to date in 20 papers. Our proband's variants were previously identified only in three independent LGMDR8 patients in homozygosis, therefore our case is the first in literature to be described as compound heterozygous for such variants. Our report also provides additional data in support of their pathogenicity, since p.(Arg394His) is currently classified as a variant of uncertain significance, while p.(Ser594Asp) as likely pathogenic. Taken together, these findings might be useful to improve both the genetic counseling and the diagnostic accuracy of this rare neuromuscular condition.


Introduction
The term limb-girdle muscular dystrophy (LGMD) refers to typically non-syndromic childhood-and adult-onset group of muscular dystrophies, affecting primarily skeletal muscles, and usually associated with elevated serum creatine kinase (CK) concentration (1,2).Patients with LGMD suffer from progressive muscle weakness and wasting, involving proximal more than distal districts, in particular muscles of the shoulder and pelvic girdles (1,2).However, other muscle groups, such as facial, distal upper and lower limbs, may also be affected (3).In the pre-molecular era, LGMD diagnosis used to be purely clinical, and it could only be confirmed differentially once specific protein testing became available (4,5) to exclude X-linked recessive neuromuscular disorders, such as Duchenne muscular dystrophy and Becker muscular dystrophy (1).Since the advent of molecular myology, pathogenic variants in 29 genes have been reported in distinct LGMD clinical presentations, classified into dominant (LGMDD) or recessive (LGMDR) forms according to the pattern of inheritance, with some genes presenting both kinds of transmission (1,2).
LGMDR8 (MIM #254110) represents the subtype 8 of the autosomal recessive type of LGMD, previously known as LGMD2H (1,2), and initially referred to as Sarcotubular Myopathy when first described in the Hutterite population of Manitoba, in North America (6)(7)(8)(9).This subtype of LGMDR can display clinical heterogeneity, with symptom onset ranging between the first and the fourth decade of life (10).The clinical course is progressive, and severity goes from absence of symptoms to muscular weakness with atrophy (11,12), possibly requiring the use of a wheelchair.

Methods
The study was approved by the institutional review board of the Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan (Italy).The patient provided written informed consent for all aspects of the study.All the details related to data collection and analysis are available in Supplementary material.Clinical, histological, immunohistochemical, MMRI, and molecular data from the proband were integrated within our literature review of additional 52 LGMDR8 patients, carrying biallelic TRIM32 variants (Table 1; Figure 1A).
As far as the literature review is concerned, the following terms were searched through PubMed in May 2023, filtering for human studies, abstract and full-text availability in English: "((trim32) AND (lgmdr8)) OR (limb-girdle muscular dystrophy recessive 8)."We included publications reporting patients of any age, and providing clinical, instrumental, and molecular characterization, with the latter clearly specifying the presence of TRIM32 biallelic variants, either carried in homozygosis or compound heterozygosis.We applied exclusion criteria at both molecular and clinical levels: (1) the identification of monoallelic TRIM32 variants led to the exclusion of the heterozygous patients; (2) among the patients carrying TRIM32 variants biallelically, we excluded those cases clearly showing a non-LGMDR8 phenotype, such as the Bardet-Biedl syndrome type 11 (BBS11).

. Case report
A 63-year-old man came to our attention presenting with progressive muscle weakness and difficulty in climbing the stairs, since the age of 39.The patient was born to non-consanguineous parents, had a typical psychomotor development, and a negative family history of neuromuscular disorders.He stopped running in 12-month time from symptom onset, developing a waddling gait and inability to walk long distances by the age of 53.Serum CK levels were persistently increased over the years, ranging between 600 and 3,000 U/L (with 350 U/L representing the upper normal limit in men); his cardiopulmonary examination was always unremarkable.
A muscle biopsy of the vastus lateralis, collected at the age of 40, revealed dystrophic changes, such as severe fibroadipose replacement, predominance of type 2 fibers, increased fiber size variability, and presence of necrotic fibers with macrophagic invasion.Furthermore, there was evidence of numerous splitting fibers, subsarcolemmal and intracytoplasmic vacuoles containing finely granular material, without the presence of sarcotubular aggregates (Figures 2A-D).Immunohistochemistry showed desmin and myotilin accumulations within muscle fibers, as well as increased p62 detection compared to control muscles (Figures 2E-G).Electromyography (EMG) and electroneurography (ENG), performed at the early stage of the disease, showed a myopathic pattern of the quadriceps femoris, supporting the diagnosis of a pelvic girdle myopathy (clinical timeline is displayed in Figure 1B).The primary hypothesis of a putative involvement of the DMD gene, possibly responsible for Becker muscular dystrophy, was ruled out by Sanger sequencing and MLPA analysis, at the time of symptom onset.
His last physical examination revealed bilateral hypotrophy of the anterior compartment of the thigh and of the gastrocnemius, winged scapula, and selective weakness of pectoral, upper and lower limb muscles, causing waddling gait and bilateral foot drop.He was able to climb the stairs and stand up from a seated position only with the use of external aids.The patient reported to have recently experienced mild weakness of the upper limbs, and right thigh myalgias, with no cramps nor myoglobinuria.He did not complain of dysphagia or dysphonia.The most recent CK dosage was 2,400 U/L, in line with his previous values.
Following the neuromuscular visit, a muscle MRI scan was performed for the first time in 24 years of disease duration.This revealed diffuse morphological changes in several muscles of the upper and lower limbs (Supplementary Figure 1), with a severe fibroadipose degeneration involving the shoulder and pelvic girdles.In particular, the fatty infiltration pattern of the pelvic girdle and of the thigh muscles showed relative sparing of rectus   Frontiers in Neurology frontiersin.orgFrontiers in Neurology frontiersin.orgfemoris, gracilis, sartorius, and the short head of biceps femoris.
The most affected compartments of the thigh were the medial and the posterior ones, with a diffuse bilateral hypotrophy, which was mostly symmetric.Dystrophic changes were also present at the level of the leg muscles, with relative sparing of the flexor hallucis longus, flexor digitorum longus and tibialis posterior muscles.As for the shoulder girdle, there was diffuse fatty degeneration, with relative sparing of the right supraspinatus, as well as of the trapezius, infraspinatus and pectoralis muscles, bilaterally.At the upper arm level, the most affected muscle was the triceps brachii, followed by the deltoid muscle.In contrast to what shown by the MMRI, clinical examination of the upper limbs was normal, with MRC score being 5 in all districts, except for pectoral muscles, where MRC score was 4 bilaterally.
To make the definitive diagnosis of LGMDR8, a Clinical Exome Sequencing was performed at our center, revealing the presence of two heterozygous TRIM32 variants (Figures 3B, C 25), and classified as class 4 (likely pathogenic) and class 3 (of uncertain significance) variants, respectively, according to the ACMG (American College of Medical Genetics and Genomics) criteria (Figure 3).Proband's parents or relatives were not available for segregation testing.
Cardiac and respiratory involvement is not a frequent presentation in LGMDR8, but there might be cardiorespiratory alterations in some cases.Cardiological examination (electrocardiography and/or echocardiography) and spirometry testing were reported normal in 77% (30/39) of the patients with available cardiorespiratory data.The remaining 23% (9/39) showed some respiratory (10%, 4/39) and/or cardiac abnormalities (21%, 8/39), such as respiratory insufficiency and violations of myocardial conduction (Table 1).
MMRI studies of the lower limbs were described in 49% (26/53) of the cases [our patient; (13,19,21,(24)(25)(26)(27)(28)].Their images showed fibroadipose degeneration, with preferential affection of the posterior thigh compartment and selective sparing of specific muscles.Upper limb MMRI findings have been rarely reported in the LGMDR8 literature [our case; (28)].Wei at al. (28) described a minimal fatty infiltration without a distinct involvement pattern; conversely, our patient's MMRI showed diffuse fibroadipose replacement of the shoulder girdle, and involvement of the high arm, with selective sparing of specific muscles.
Histological findings were reported in 39 patients: 69% (27/39) of the biopsies showed nonspecific myopathic changes, whereas the remaining 31% (12/39) displayed a clearly dystrophic pattern, with two cases presenting both myopathic and neurogenic features.Immunohistochemical analysis of our patient's biopsy showed accumulations of desmin and myotilin, that are muscle-relevant targets of the TRIM32 ubiquitin ligase activity, alongside dysbindin, actin, α-actinin and tropomyosin (14).Similar findings were also observed in another LGMDR8 patient (25), who was homozygous for the c.1781G > A, p.(Ser594Asn) TRIM32 variant, also carried by our proband.Furthermore, a certain degree of autophagy alteration has been observed in muscle samples from LGMDR8 patients: reduced levels of p62 and LC3II were described by Servián-Morilla et al. (26), whereas an increased p62 signal was detected in the muscle biopsy of our patient.Further investigations are therefore needed to elucidate TRIM32 pathways in autophagy regulation, and whether autophagy is up-or down-regulated in LGMDR8 (14).

Discussion
The TRIM family includes RING E3 ubiquitin ligases sharing a common evolutionary origin and a similar structure of the Nterminal RING domain as well as of the NHL repeats at the Cterminus.
Looking at the clinical features and genotypes of the 53 patients from our literature review, a clear genotype-phenotype correlation did not emerge.Interestingly, Guan and coauthors (20) recently analyzed 86 TRIM32-mutated patients from the literature, highlighting a correlation of the TRIM32 variants exclusively impacting on the NHL domains, with a lower age at onset and higher CK levels, compared to subjects carrying variants outside the NHL regions.A gender effect was also observed, with a lower age of symptom onset, and higher CK levels, in males than in females (20).However, their analysis included both LGMDR8 and BBS11 patients, carrying bi-or monoallelic TRIM32 variants (20), and this may explain the different results from our review, which instead specifically focused on LGMDR8-associated TRIM32 biallelic variants.
To date, 50% (13/26) of the TRIM32 variants (Table 1) considered to be causative for LGMDR8 (when present biallelically, either in homozygosis or in compound heterozygosis) are located in one of the five NHL repeats, like in the case of our patient.However, LGMDR8-causing TRIM32 variants have been recently identified also in non-NHL domains (RING and Coiled-Coil), as well as in the intermediate regions outside the structural domains, like the pre-RING region (19), inter-CoiledCoil-NHL1 (26), and inter-NHL3-NHL4 (21).These findings dismantled the initial hypothesis of a selective involvement of the NHL domains in the LGMDR8 pathogenesis (14).In particular, the RING domain was found to be mutated (p.Cys20Phe, p.Cys39Leufs  (22).The only TRIM32 structural domain which seems not to be involved in the LGMDR8 pathogenesis is the B-box-2.To date, the only noted B-box-2 variant is p.(Pro130Ser), which is, in fact, associated with BBS11 (33).BBS11 is a multisystemic disorder with no skeletal muscle involvement, characterized by obesity, polydactyly, retinal dystrophy and kidney abnormalities.Interestingly, despite LGMDR8 and BBS11 being two distinct disorders, some of the LGMDR8 patients (26) carrying the truncating variant, p.Cys39Leufs * 17, in the RING domain, showed typical BBS11 systemic symptoms in addition to the LGMDR8 muscular presentation.A putative explanation for the presence of such different TRIM32-related phenotypes might be that RING and B-Box-2 domains have less muscular specificity and a more pleiotropic activity than the Coiled-Coil domain and the NHL repeats, that instead seem to mediate more musclespecific functions.
Our patient carried a likely pathogenic variant in the NHL1 domain, and a variant of uncertain significance in the NHL5 domain.The NHL1 variant, p.(Arg394His), was previously reported in homozygosis in two LGMDR8 patients by Saccone et al. (11) and Johnson et al. (21); whereas the NHL5 variant, p.(Ser594Asp), was described in homozygosis in one patient by Panicucci et al. (25).Clinical, histological, and imaging features of our proband were very similar to the ones described in the patient carrying the NHL5 variant (25).As for the other two cases carrying the same NHL1 variant harbored by our patient, one had analogous muscular presentation and progressiveness of the disease (21), whereas the second patient, unlike our proband, presented with additional cardiorespiratory symptoms (11).Interestingly, two of these three cases were of Italian origins (11,25), like our patient.

Conclusions
We presented a novel LGMDR8 patient of Italian origins, caused by two TRIM32 missense variants for the first time described in compound heterozygosis, and previously reported in homozygosis in three independent patients (11,21,25).This case report provided new evidence in support of the pathogenicity of both p.(Arg394His) and p.(Ser594Asp) variants, that are not yet classified as pathogenic.We also defined the typical LGMDR8 phenotype associated with the so far identified 26 TRIM32 variants, by performing a literature review of clinical and instrumental data related to 53 LGMDR8 patients.All these findings, taken together, might potentially be of help in improving both the diagnostic accuracy and the genetic counseling of this rare neuromuscular disease.

FIGURE( A )
FIGURE (A) Graphic of the typical LGMDR phenotype.The five sections summarize the most frequent clinical and instrumental features, drawn from the analysis of theLGMDR TRIM -mutated patients (including our case) reported in the literature; (B) timeline highlighting our patient's clinical history and diagnostic work-up.Figures (A, B) were created with BioRender.com.

FIGURE
FIGURE Histological and immunohistochemical findings in muscle biopsy.(A, B) Hematoxylin and eosin (A) and modified Gomori trichrome (B) stains showing markedly increased fiber size variability, central nuclei, severe fibro-adipose replacement (asterisks), splitting fibers (arrowheads), internal vacuoles (arrow).(C) ATPase pH .stain showing predominant type (darker) fibers atrophy.(D) NADH-TR stain showing preserved oxidative enzymes activity in most of the observed fibers, with darker stain of the atrophic fibers.(E, F) Immunofluorescence assay showing increased intracellular accumulation of myotilin (E) and desmin (F) proteins compared to control muscles (small frames).(G) Immmunofluorescence assay showing increased cytoplasmic p signal compared to control muscle (small frame).Scale bars: µm (A-D) µm (E-G).

FIGURE
FIGURE Schematic representation of the TRIM gene (A) and TRIM protein (B), displaying the structural domains and intermediate regions with the LGMDR -associated biallelic variants (that are listed with the same color coding in Table ).The two variants (c.G > A, p.Arg His; c. G > A, p.Ser Asn) carried by our patient are outlined in red (B) and also shown in the chromatograms (C).Figures (A, B) were created with BioRender.com.
): c.1181G > A/p.(Arg394His), and c.1781G > A/p.(Ser594Asp).These were already described by Saccone et al. (11) and Panicucci et al. ( TABLE Summary of the TRIM variants carried biallelically by the LGMDR patients (including our case) reported in the literature.