TRAPPC11 and GOSR2 mutations associate with hypoglycosylation of α-dystroglycan and muscular dystrophy

TRAPPC11 and GOSR2 mutations associate with hypoglycosylation of α-dystroglycan and muscular... Background: Transport protein particle (TRAPP) is a supramolecular protein complex that functions in localizing proteins to the Golgi compartment. The TRAPPC11 subunit has been implicated in muscle disease by virtue of homozygous and compound heterozygous deleterious mutations being identified in individuals with limb girdle muscular dystrophy and congenital muscular dystrophy. It remains unclear how this protein leads to muscle disease. Furthermore, a role for this protein, or any other membrane trafficking protein, in the etiology of the dystroglycanopathy group of muscular dystrophies has yet to be found. Here, using a multidisciplinary approach including genetics, immunofluorescence, western blotting, and live cell analysis, we implicate both TRAPPC11 and another membrane trafficking protein, GOSR2, in α-dystroglycan hypoglycosylation. Case presentation: Subject 1 presented with severe epileptic episodes and subsequent developmental deterioration. Upon clinical evaluation she was found to have brain, eye, and liver abnormalities. Her serum aminotransferases and creatine kinase were abnormally high. Subjects 2 and 3 are siblings from a family unrelated to subject 1. Both siblings displayed hypotonia, muscle weakness, low muscle bulk, and elevated creatine kinase levels. Subject 3 also developed a seizure disorder. Muscle biopsies from subjects 1 and 3 were severely dystrophic with abnormal immunofluorescence and western blotting indicative of α-dystroglycan hypoglycosylation. Compound heterozygous mutations in TRAPPC11 were identified in subject 1: c.851A>C and c.965+5G>T. Cellular biological analyses on fibroblasts confirmed abnormal membrane trafficking. Subject 3 was found to have compound heterozygous mutations in GOSR2: c.430G>T and c.2T>G. Cellular biological analyses on fibroblasts from subject 3 using two different model cargo proteins did not reveal defects in protein transport. No mutations were found in any of the genes currently known to cause dystroglycanopathy in either individual. Conclusion: Recessive mutations in TRAPPC11 and GOSR2 are associated with congenital muscular dystrophy and hypoglycosylation of α-dystroglycan. This is the first report linking membrane trafficking proteins to dystroglycanopathy and suggests that these genes should be considered in the diagnostic evaluation of patients with congenital muscular dystrophy and dystroglycanopathy. Keywords: TRAPPC11, GOSR2, Golgi, Dystroglycanopathy, Dystroglycan, Muscular dystrophy, Glycosylation, Membrane traffic * Correspondence: michael.sacher@concordia.ca; steven-moore@uiowa.edu Austin A. Larson, Peter R. Baker II, Miroslav P. Milev, Michael Sacher and Steven A. Moore contributed equally to this work. Department of Biology, Concordia University, Montreal, Canada Department of Pathology Carver College of Medicine, The University of Iowa, Iowa City, IA, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Larson et al. Skeletal Muscle (2018) 8:17 Page 2 of 10 Background L. She had significant regression of development with Dystroglycanopathies are a group of muscular dystrophies loss of rolling and sitting, loss of fine motor and verbal resulting from abnormal glycosylation of α-dystroglycan skills, and inability to feed orally after this illness. (α-DG) that leads to reduced binding affinity for extracel- MRI of the lower extremities showed high signal on lular matrix proteins [1]. The clinical phenotypes span a short tau inversion recovery (STIR) sequences of the broad range from the congenital muscular dystrophies deep and superficial posterior compartments bilaterally (CMDs) with brain and eye malformations to adult-onset (Fig. 1c). Skeletal muscle and liver biopsies were limb-girdle muscular dystrophy (LGMD) [2]. Dystroglycan obtained at 9 months of age. Liver biopsy showed micro- is encoded by DAG1 and is cleaved into α-DG and β-DG vesicular steatosis (Fig. 1d, e). Skeletal muscle showed an after translation [3]. DAG1 is widely expressed in different active dystrophic process (Fig. 1g) and hypoglycosylation human tissues, consistent with the multi-organ pheno- of α-DG by both immunofluorescence and western blot- types of many individuals with the most severe forms of ting (Fig. 2). In contrast, α-DG in cultured fibroblasts was dystroglycanopathy [4]. indistinguishable from control fibroblasts in on-cell and in Mutations in DAG1 itself as well as 17 other genes WGA glycoprotein western blots (data not shown). This is have been reported in patients with dystroglycanopathy. not uncommon and has been reported in the case of other These include glycosyltransferases (POMT1, POMT2, genes involved in dystroglycanopathy [6, 7]. POMGNT1, POMGNT2, B3GALNT2, B3GNT1, LARGE, The patient had several subsequent acute infectious TMEM5), a kinase (POMK), five genes encoding en- illnesses with seizures and neurological regression. zymes necessary for dolichol-P-mannose (dol-P-man) Follow-up brain MRI at 15 months of age showed synthesis (DOLK, DPM1, DPM2, DPM3, and GMPPB), marked progressive volume loss (Fig. 1b). Glycosylation and three genes encoding proteins necessary for joining analysis of transferrin and ApoCIII proteins in serum by the α-DG-linked core glycan structure with the distal affinity chromatography-mass spectrometry (Mayo ligand-binding region of the structure via a ribitol Medical Laboratory) as well as by MALDI-TOF mass phosphate disaccharide (FKTN, FKRP, ISPD)[5]. To spectrometry (Emory Genetics Laboratory) was normal. date, no membrane trafficking proteins have been impli- In her last evaluation, at 3 years and 6 months of age, cated in dystroglycanopathies. she was areflexic with limited antigravity strength and In this study, we report clinical, histopathological, severe hypotonia. She fed exclusively via gastrostomy biochemical, and molecular genetic data on two families tube with no verbal communication. Seizures were well with CMD and hypoglycosylation of α-DG. Two genes, controlled on levetiracetam monotherapy. CK remained TRAPPC11 and GOSR2, that each have a role in mem- elevated with values as high as 19,000 U/L. She had mild brane trafficking in the biosynthetic pathway have been hepatomegaly and aminotransferases were still signifi- implicated as candidate dystroglycanopathy genes. They cantly elevated with 4:1 ALT to AST ratio, but there was represent the first membrane trafficking proteins impli- no coagulopathy or hyperbilirubinemia. She chronically cated in α-DG hypoglycosylation. Since TRAPPC11 required noninvasive positive pressure ventilation with mutations have been reported in a number of individuals sleep. After multiple hospital admissions for viral suffering from a muscular dystrophy, and these individ- respiratory infections, she underwent immunological uals also display membrane trafficking defects in cul- evaluation and was found to have impaired natural killer tured fibroblasts, this gene should be considered in the cell function on multiple repeated analyses. She did not diagnostic evaluation of patients with CMD. have peripheral neuropathy, cataracts, alacrima, achala- sia, renal disease, hearing loss, or cholestasis. Case presentation Exome trio sequencing showed compound heterozy- Family 1 gous rare variants in trans in TRAPPC11 (NM_021942): Subject 1 presented with status epilepticus in the setting c.851A>C (p.Q284P) and c.965+5G>T (intron 9 splice of a vomiting illness at 6 months of age. Magnetic reson- site disruptor). The p.Q284P missense mutation was ance imaging (MRI) of the brain showed bilateral multi- absent from the Exome Aggregation Consortium (ExAC) focal restricted diffusion of the cortex, the cerebral white database, and c.965+5G>T was present in 2/119,770 matter, and the pons (Fig. 1a). Her serum aminotransfer- alleles [8]. The latter mutation resulted in a transcript ases were elevated with alanine aminotransferase (ALT) that lacks exon 9 and the first 88 bases of exon 10 of ~ 1600 U/L and aspartate aminotransferase (AST) ~ (Fig. 3a) and is predicted to result in an in-frame dele- 400 U/L as well as a prolonged prothrombin time of tion of amino acids 278–351 (p.I278_Q351del). Cultured 20.7 s (normal range is 12–15 s), consistent with syn- fibroblasts had greatly reduced levels of TRAPPC11 thetic liver dysfunction. The approximately 4:1 ALT to (Fig. 3b) suggesting the p.Q284P protein and the pre- AST ratio was consistent over multiple measurements. dicted p.I278_Q351del protein are unstable. These fibro- Creatine kinase (CK) at initial presentation was 3500 U/ blasts showed a delay in the maturation of the marker Larson et al. Skeletal Muscle (2018) 8:17 Page 3 of 10 Fig. 1 Subjects 1 and 3 display brain, liver, and muscle abnormalities. a Diffusion-weighted (B1000) MRI showing restricted diffusion of the medial occipital cortex and underlying white matter at 6 months of age in subject 1 at the time of initial presentation. b Fluid-attenuated inversion recovery (FLAIR) MRI for subject 1 at 15 months notable for marked cerebral volume loss. c Short tau inversion recovery (STIR) shows symmetric high signal in the posterior compartments of the legs of subject 1 at 12 months of age. Subject 1 has microvesicular steatosis of the liver; light microscopy hematoxylin and eosin (d) and electron microscopy (e). Note the lipid accumulations marked by the arrows in e. f–h Muscle biopsies from control (f), subject 1 (g), and subject 3 (h) were stained with hematoxylin and eosin. Dystrophic features are present in subjects 1 and 3. The size bar denotes 50 μmin d and f–h. The size bar denotes 5 μmin e protein VSVG-GFP ts045 (Fig. 3c, d). Analysis of 9 months, she was areflexic. She had low muscle bulk live-cell trafficking revealed a delay in the release of and myopathic facial appearance and did not have anti- VSVG-GFP ts045 from the Golgi (Fig. 3e, f) as well as a gravity strength. She had fine nystagmus but otherwise delay in arrival of a Golgi marker (sialyl intact extraocular movements. MRI of the brain was transferase-SBP-GFP) from the endoplasmic reticulum normal at ages 2 and 4 years. She died due to respiratory (Fig. 3g, h). The delayed release of protein from the failure at age 5 years. Golgi is consistent with the initial findings reported by Subject 3 is the younger sister of subject 2. She was Bögershausen et al. in LGMD2S patients with noted to have muscle weakness and hypotonia at TRAPPC11 mutations [9], and the delayed arrival of 3 months of age. At 9 months, she had antigravity protein to the Golgi is consistent with the findings of strength only. Serum CK value was 1760 U/L. At Scrivens et al. [10]. 19 months, skeletal muscle biopsy was obtained showing an active dystrophic process (Fig. 1h) and hypoglycosyla- Family 2 tion of α-DG by both immunofluorescence and western Subject 2 presented for medical evaluation at age blotting (Fig. 2). In contrast, α-DG in cultured fibro- 6 months for hypotonia. She was found to have CK blasts was indistinguishable from control fibroblasts in values of up to ~ 5000 U/L. She developed absence on-cell and in WGA glycoprotein western blots (data seizures at age 2 years. She had steadily progressive not shown). Furthermore, the VSVG-GFP membrane muscle weakness. On examination at age 4 years and trafficking assay kinetics as well as arrival of the Golgi Larson et al. Skeletal Muscle (2018) 8:17 Page 4 of 10 Fig. 2 Subjects 1 and 2 display abnormalities in both α-dystroglycan staining and glycosylation. Control muscle or muscle taken from subject 1 (S1) and subject 3 (S3) were stained for alpha dystroglycan using VIA4-1 antibody (a)or β-DG (b). Note the reduced staining for α-DG but not β-DG in subjects 1 and 3. The size bar denotes 50 μm for all panels in a and b. c Western blot analysis of muscle tissue from control and subjects 1 and 3. Samples were probed with peptide-specific antibody AF6868 and the glycoepitope-specific antibody IIH6 as indicated. The location of α-DG and β-DG is indicated. Note that control shows a higher molecular size immunoreactive species for α-DG with both antibodies while S1 and S3 show a more heterogeneous species of much smaller molecular size, suggesting hypoglycosylation of the protein marker was indistinguishable from control fibroblasts Physical exam revealed findings similar to her sister. (Fig. 3c–f). Apart from her sister, there is no family history of At age 2.5 years, she developed a seizure disorder neuromuscular disease. The subject is now 6 years of characterized as focal seizures but later as both focal and age with medically refractory epilepsy and progressive generalized, which often became intractable and re- severe muscle weakness. Clinical exome trio sequencing quired hospitalization. Evaluation showed no evidence of was performed, and no relevant sequence variants were nystagmus and ocular range of motion was full. There initially reported. In a targeted sequencing panel, subject were no focal deficits and her cranial nerves were nor- 3 was found to have compound heterozygous rare vari- mal. She demonstrated severe weakness and muscular ants in GOSR2 (NM_001012511): c.430G>T (p.G144W) hypotonia. MRI of the brain showed diffuse volume loss and c.2T>G. Retrospective evaluation of GOSR2 in the resulting in ex vacuo ventriculomegaly. EEG at 2 years whole exome sequencing (WES) data confirmed that and 7 months of age showed runs of spike and wave both variants were present in subject 3 and were in discharges originating in the occipital lobe which were trans. Extensive re-evaluation of seizure and dystrogly- exacerbated by photic stimuli. Head circumference was canopathy loci in the WES failed to identify any other at the 30th centile, height at the 10th centile, and weight pathologic variants. The GOSR2 p.G144W missense below the 1st centile. variant is a previously reported disease-causing muta- At 3.5 years of age, she developed episodes of vomiting tion and is present in 5/121,408 alleles in the ExAC and apparent abdominal pain. This led to the detection database with no homozygous individuals. The second of elevated ALT of up to 700 U/L. An extensive evalu- variant (c.2T>G) is present in 1/18,808 alleles in the ation for infectious, anatomical, autoimmune, and meta- ExAC database [8]. The mutation is likely to result in bolic etiologies of liver disease was nondiagnostic. the use of an alternate start codon with elimination of Larson et al. Skeletal Muscle (2018) 8:17 Page 5 of 10 Fig. 3 (See legend on next page.) Larson et al. Skeletal Muscle (2018) 8:17 Page 6 of 10 (See figure on previous page.) Fig. 3 TRAPPC11 compound heterozygous mutations affect membrane trafficking in patient fibroblasts. a mRNA was collected from control and subject 1 (S1), converted to cDNA and amplified by PCR using oligonucleotides annealing to exons 8 and 11. The amplicons were sequenced and found to represent exons 8-9-10-11 (higher molecular size amplicon) and exons 8-part of 10-11 (lower molecular size amplicon). b Lysates from control and subject 1 (S1) fibroblasts were probed for TRAPPC11 and tubulin as a loading control. c Fibroblasts were infected with VSVG- GFP ts045, and the protein was arrested in the endoplasmic reticulum (ER) by shifting the cells to 40 °C. The protein was synchronously released from the ER upon downshifting the temperature to 32 °C, and the acquisition of Endoglycosidase H (EndoH) resistance was assayed at the times indicated. A representative western blot is displayed, and quantification of a minimum of three such blots is shown in d. e The same assay as in b was performed on live cells and the arrival and release of the GFP signal was quantified over time. Representative images from the movies are displayed in e, and quantification of the signal in the Golgi region is shown in f. To more accurately measure ER-to-Golgi trafficking, the RUSH assay [36] was performed using ST-SBP-GFP with the Ii hook (g). Images were acquired over time in live cells upon addition of biotin to initiate release of the protein from the ER. Quantification of the signal in the Golgi is displayed in h. Size bars in e and g denote 25 μm. Error bars represent SEM from a minimum of three replicates in d. N values for f and h are indicated in the figure 18 amino acids from the amino-terminus of the protein disease is a disorder of glycosylation, analysis of glycoepi- according to MutationTaster2 and is presumed to be topes of secreted proteins may not be a sensitive test for pathogenic as a result [11]. diagnostic purposes. The zebrafish model of TRAPPC11-related disease shows Discussion and conclusions generalized impairment of N-linked glycosylation as well as In this report, we show that mutations in two genes depletion of lipid-linked oligosaccharides (LLOs) [17]. The encoding proteins involved in membrane trafficking, inability to synthesize dolichol-P-mannose (dol-P-man), a TRAPPC11 and GOSR2, are associated with CMD and lipid-linked saccharide, is a known cause of dystroglycano- dystroglycanopathy. Biallelic mutations in TRAPPC11 pathy [7]. Expression of multiple glycosylation-related genes were initially reported as the etiology of LGMD2S in (including the known etiologies of dystroglycanopathy 2013 [9] and have since been associated with a variety of gmppb, dpm1, dpm2,and dpm3) showed significant com- multisystemic phenotypic findings including intellectual pensatory upregulation in the trappc11 fish [17]. Interest- disability, seizures, microcephaly, cerebral atrophy, ingly, TRAPPC11 siRNA knockdown in HeLa cells had a cataracts, alacrima, achalasia, hepatic steatosis, and specific inhibitory effect on glycosylation that was not cholestatic liver disease, in addition to muscular present with knockdown of other components of the dystrophy [9, 12–15]. Comparisons between subject 1 TRAPP complex. This led to the conclusion that and all published mutations in TRAPPC11 and associ- TRAPPC11 may have another function that is independent ated phenotypes are summarized in Table 1. Our study of its role in vesicle transport and led to speculation that now adds two new mutations with functional validation impaired LLO synthesis may be the most relevant function and categorizes TRAPPC11-related disease as a of TRAPPC11 in the process of protein glycosylation [17]. dystroglycanopathy. Finally, trappc11 zebrafish mutations were shown to lead to TRAPPC11 dysfunction may contribute to disease fatty liver via a pathological activation of the unfolded pathophysiology in several ways. Extensive functional protein response. This may be relevant to subject 1 as well studies of cultured fibroblasts were conducted by as the other reported individuals with hepatopathy and Bögershausen et al. [9]. They demonstrated that cells TRAPPC11-related disease [13]. Taken together, several had abnormally fragmented and diffuse Golgi; delayed mechanisms for the role of TRAPPC11 in muscular and traffic out of the Golgi and the proteins LAMP1 and hepatic phenotypes are known and can explain many LAMP2 were found to be abnormally glycosylated. clinical features of subject 1. TRAPP (transport protein particle) forms several related Human mutations in GOSR2 were first reported in multisubunit trafficking complexes (MTCs) that partici- 2011 in six individuals with the same homozygous pate in the tethering of vesicles to target membranes, missense mutation (c.430G>T) who had progressive including vesicles associated with the Golgi [10]. Since myoclonus epilepsy (PME), ataxia, scoliosis, and mildly the Golgi is the major site of protein glycosylation in the elevated serum CK (see Table 1 for a comparison cell [16], defects in Golgi morphology and traffic can between subjects 2 and 3 with all reported GOSR2 muta- result in protein glycosylation defects. Recently, abnor- tions) [18]. All individuals were areflexic in early child- mal glycosylation of serum transferrin was described in hood and were non-ambulatory by adolescence or early a patient with compound heterozygous mutations in adulthood. Muscle histology and EMG were normal. An TRAPPC11, consistent with a type 2 disorder of glyco- additional eleven individuals with similar clinical presen- sylation [12]. We were unable to detect abnormalities in tations and the same homozygous mutation were glycosylation of serum transferrin using two different com- reported in 2013 and 2014 [19, 20]. The maximum CK monly employed methods. Thus, while TRAPPC11-related value reported in any of the patients was 2467 U/L. Larson et al. Skeletal Muscle (2018) 8:17 Page 7 of 10 Table 1 Comparison of all known TRAPPC11 and GOSR2 mutations Genotype Number of Neurological phenotype Muscle phenotype Other features References cases TRAPPC11 3 motor delay in one individual, otherwise LGMD, CK up to ~ 2800 scoliosis, cataracts and esotropia Bogershausen et al. [9] c.2938G>A normal each in one individual c.2938G>A TRAPPC11 5 epilepsy, developmental delay, ataxia, myopathy, CK up to ~ 1200 short stature, exophoria in one individual Bogershausen et al. [9] c.1287+5G>A chorea, microcephaly, cerebral atrophy c.1287+5G>A TRAPPC11 4 developmental delay, cerebral atrophy, CMD, CK not reported, dystrophic scoliosis, achalasia, alacrima Koehler et al. [14] c.1893+3A>G medically refractory epilepsy appearance of biopsied muscle tissue c.1893+3A>G TRAPPC11 2 moderate intellectual disability, ambulatory, CMD, CK up to ~ 10,000; dystrophic cataracts, significantly elevated ALT, mildly Fee et al. [15] c.513_516delTTTG seizures, MRI with mild atrophy biopsied muscle, abnormal dystroglycan elevated AST, liver fibrosis c.2330A>C staining TRAPPC11 1 developmental delay, decreased white CMD, CK up to ~ 9000; abnormal signal hepatic steatosis, significantly elevated ALT, Liang et al. [13] c.2938G>A matter volume on MRI in posterior compartment leg muscles mildly elevated AST, cataracts c.661-1G>T on CT scan TRAPPC11 1 microcephaly, brain atrophy on MRI, presumed CMD, hypotonia, CK not retrognathia, cholestatic liver disease, Matalonga et al. [12] c.1141C>G sensorineural hearing loss, peripheral reported thrombocytopenia, nephropathy, osteopenia c.3310A>G neuropathy TRAPPC11 1 severe developmental delay, multifocal CMD, CK up to ~ 18,000; abnormal signal hepatic steatosis, significantly elevated ALT, This paper c.851A>C restricted diffusion on MRI; later cerebral in posterior compartment leg muscles on mildly elevated AST; retinopathy, impaired c.965+5G>T atrophy MRI scan; dystrophic appearance of biopsied NK cell function muscle; hypoglycosylation of α-dystroglycan GOSR2 17 “North Sea” progressive myoclonus epilepsy; CK up to ~ 2500 but normal in some; no scoliosis, pes cavus, syndactyly in some, Lomax et al. [19], Egmond c.430G>T childhood-onset ataxia, loss of ambulation specific abnormalities reported in muscle delayed puberty in some et al. [20], Corbett et al. [18] c.430G>T in early adulthood biopsies GOSR2 1 progressive myoclonus epilepsy, ataxia; MRI none reported none reported Praschberger et al. [37] c.430G>T with cerebral atrophy c.491_493delAGA GOSR2 2 medically refractory epilepsy; MRI with CMD, CK up to ~ 5000; dystrophic muscle no additional findings This paper c.430G>T cerebral atrophy biopsy with hypoglycosylation of c.2T>G α-dystroglycan; severe weakness and respiratory failure leading to death at 5 years in older sibling Larson et al. Skeletal Muscle (2018) 8:17 Page 8 of 10 There was no specific assessment of α-DG glycosylation addition of ribitol phosphate molecules to link the core in their muscle biopsies. Subjects 2 and 3 in our study and ligand-binding regions of the α-DG glycan structure have a much more severe phenotype. Since CMD [33–35]. Our study suggests that TRAPPC11 and represents the severe end of the clinical spectrum of GOSR2 are also involved in the trafficking and glycosyla- GOSR2-related disease and PME represents the milder tion of dystroglycan in the Golgi. This represents the end of the spectrum, the new c.2T>G mutation resulting first report of an association between these genes and in CMD reported in our study likely cause more severe α-DG hypoglycosylation. It remains to be seen if other perturbation of Golgi function than the common GOSR2 mutations associate with similar cellular and c.430G>T mutation. It remains unclear which aspect of clinical phenotypes. Given the number of individuals with Golgi function is affected since a membrane trafficking TRAPPC11 mutation-associated muscular dystrophy, it may defect in neither the VSVG-GFP marker protein nor a be prudent for this gene to now be considered in the diag- resident Golgi enzyme was detected. Future studies nostic evaluation of patients with dystroglycanopathy. should examine the trafficking of Golgi-localized Abbreviations glycosyl transferases that are responsible for α-DG ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; CK: Creatine processing. kinase; FLAIR: Fluid-attenuated inversion recovery; MRI: Magnetic resonance imaging; STIR: Short tau inversion recovery; α-DG: α-Dystroglycan GOSR2 encodes a Golgi Qb-SNARE (soluble N-ethyl- maleimide-sensitive factor attachment protein receptor) Acknowledgements protein. In the cell, GOSR2 localizes to the cis-Golgi and We are grateful to members of our laboratories for constructive comments on this report and for fruitful discussions. mediates docking and fusion of vesicles originating from the ER. There is precedent for Golgi dysfunction leading Funding to diseases manifesting with abnormal glycosylation and SAM and MOC are funded by the Iowa Wellstone Muscular Dystrophy Cooperative Research Center U54, NS053672. MS is funded by the Canadian multisystemic disease. Examples include the disease Institutes of Health Research and the Natural Sciences and Engineering caused by mutations in genes that encode the COG Research Council of Canada. RJS is funded by NCATS/NIH, UL1TR001082. (conserved oligomeric Golgi) complex, an MTC that Availability of data and materials localizes to the Golgi [21]. Additionally, an individual All data generated or analyzed during this study are included in this has been described with CMD due to homozygous mu- published article. tations in GOLGA2, a golgin protein that also impacts Authors’ contributions Golgi function [22]. The potential for a link between AAL designed the study and drafted the manuscript. PRB II designed the aberrant Golgi trafficking and dystroglycanopathy stems study and edited the manuscript. MPM performed the cell biological from an experiment employing a modified virus that membrane trafficking assays and edited the manuscript. CAP and RJS edited the manuscript. MOC grew the fibroblast cultures and performed western required normally glycosylated α-DG for cell entry. blots. JKL, AAS, and ADB developed the dystroglycanopathy sequencing Knockouts of known dystroglycanopathy genes in panel and performed genetic evaluation of subject 3. JMM evaluated the cultured fibroblasts resulted in impaired viral cell entry. sequencing data and provided a muscle biopsy for subject 3. KP performed the biochemical membrane trafficking assay. TFT analyzed the splicing defect Among the other knockouts shown to impair viral cell in subject 1. CAW evaluated the subject 3. MS designed the study and entry were those cells with mutations in several of the edited the manuscript. SAM designed the study, evaluated the muscle COG complex genes [23]. biopsy and cultured fibroblasts, coordinated the clinical groups, and edited the manuscript. All authors read and approved the final manuscript. Dystroglycanopathies result in muscular dystrophy due to dysfunctional linkage of the sarcolemma to the extra- Ethics approval and consent to participate cellular matrix. This linkage occurs via α-DG and relies All studies were completed according to local ethical approval of the institutional review boards. All individuals or their guardians gave written on the synthesis of a complex LARGE-glycan for normal informed consent before undergoing evaluation and testing, in agreement function [5]. Since the initial descriptions of dystroglyca- with the Declaration of Helsinki and approved by the ethical committees of the nopathy [1, 24–26], a variety of molecular mechanisms centers participating in this study, where biological samples were obtained. of the diseases have been discovered. Specific glycosyl- Consent for publication transferases such as POMT1/POMT2 are required to Consent for publication has been obtained by the participants or their legal construct the core glycan structure that is linked to guardians. α-DG [25, 26]. Mutations in DOLK, DPM1, DPM2, Competing interests DPM3, and GMPPB likely lead to a deficiency of The authors declare that they have no competing interests. dol-P-man (a lipid-linked monosaccharide) resulting in abnormal N-linked glycosylation as well as the O-linked Publisher’sNote mannosylation defect that results in dystroglycanopathy Springer Nature remains neutral with regard to jurisdictional claims in [6, 7, 27–31]. 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Kanagawa M, Kobayashi K, Tajiri M, Manya H, Kuga A, Yamaguchi Y, Milev MP, Stanga D, Kadakia D, et al. trappc11 is required for protein Akasaka-Manya K, Furukawa J-I, Mizuno M, Kawakami H, et al. Identification glycosylation in zebrafish and humans. Mol Biol Cell. 2016;27:1220–34. of a post-translational modification with ribitol-phosphate and its defect in 18. Corbett MA, Schwake M, Bahlo M, Dibbens LM, Lin M, Gandolfo LC, Vears muscular dystrophy. Cell Rep. 2016;14:2209–23. DF, O'Sullivan JD, Robertson T, Bayly MA, et al. A mutation in the Golgi Qb- Larson et al. Skeletal Muscle (2018) 8:17 Page 10 of 10 36. Boncompain G, Divoux S, Gareil N, de Forges H, Lescure A, Latreche L, Mercanti V, Jollivet F, Raposo G, Perez F. Synchronization of secretory protein traffic in populations of cells. Nat Methods. 2012;9:493–8. 37. Praschberger R, Balint B, Mencacci NE, Hersheson J, Rubio-Agusti I, Kullmann DM, Bettencourt C, Bhatia K, Houlden H. 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Abstract

Background: Transport protein particle (TRAPP) is a supramolecular protein complex that functions in localizing proteins to the Golgi compartment. The TRAPPC11 subunit has been implicated in muscle disease by virtue of homozygous and compound heterozygous deleterious mutations being identified in individuals with limb girdle muscular dystrophy and congenital muscular dystrophy. It remains unclear how this protein leads to muscle disease. Furthermore, a role for this protein, or any other membrane trafficking protein, in the etiology of the dystroglycanopathy group of muscular dystrophies has yet to be found. Here, using a multidisciplinary approach including genetics, immunofluorescence, western blotting, and live cell analysis, we implicate both TRAPPC11 and another membrane trafficking protein, GOSR2, in α-dystroglycan hypoglycosylation. Case presentation: Subject 1 presented with severe epileptic episodes and subsequent developmental deterioration. Upon clinical evaluation she was found to have brain, eye, and liver abnormalities. Her serum aminotransferases and creatine kinase were abnormally high. Subjects 2 and 3 are siblings from a family unrelated to subject 1. Both siblings displayed hypotonia, muscle weakness, low muscle bulk, and elevated creatine kinase levels. Subject 3 also developed a seizure disorder. Muscle biopsies from subjects 1 and 3 were severely dystrophic with abnormal immunofluorescence and western blotting indicative of α-dystroglycan hypoglycosylation. Compound heterozygous mutations in TRAPPC11 were identified in subject 1: c.851A>C and c.965+5G>T. Cellular biological analyses on fibroblasts confirmed abnormal membrane trafficking. Subject 3 was found to have compound heterozygous mutations in GOSR2: c.430G>T and c.2T>G. Cellular biological analyses on fibroblasts from subject 3 using two different model cargo proteins did not reveal defects in protein transport. No mutations were found in any of the genes currently known to cause dystroglycanopathy in either individual. Conclusion: Recessive mutations in TRAPPC11 and GOSR2 are associated with congenital muscular dystrophy and hypoglycosylation of α-dystroglycan. This is the first report linking membrane trafficking proteins to dystroglycanopathy and suggests that these genes should be considered in the diagnostic evaluation of patients with congenital muscular dystrophy and dystroglycanopathy. Keywords: TRAPPC11, GOSR2, Golgi, Dystroglycanopathy, Dystroglycan, Muscular dystrophy, Glycosylation, Membrane traffic * Correspondence: michael.sacher@concordia.ca; steven-moore@uiowa.edu Austin A. Larson, Peter R. Baker II, Miroslav P. Milev, Michael Sacher and Steven A. Moore contributed equally to this work. Department of Biology, Concordia University, Montreal, Canada Department of Pathology Carver College of Medicine, The University of Iowa, Iowa City, IA, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Larson et al. Skeletal Muscle (2018) 8:17 Page 2 of 10 Background L. She had significant regression of development with Dystroglycanopathies are a group of muscular dystrophies loss of rolling and sitting, loss of fine motor and verbal resulting from abnormal glycosylation of α-dystroglycan skills, and inability to feed orally after this illness. (α-DG) that leads to reduced binding affinity for extracel- MRI of the lower extremities showed high signal on lular matrix proteins [1]. The clinical phenotypes span a short tau inversion recovery (STIR) sequences of the broad range from the congenital muscular dystrophies deep and superficial posterior compartments bilaterally (CMDs) with brain and eye malformations to adult-onset (Fig. 1c). Skeletal muscle and liver biopsies were limb-girdle muscular dystrophy (LGMD) [2]. Dystroglycan obtained at 9 months of age. Liver biopsy showed micro- is encoded by DAG1 and is cleaved into α-DG and β-DG vesicular steatosis (Fig. 1d, e). Skeletal muscle showed an after translation [3]. DAG1 is widely expressed in different active dystrophic process (Fig. 1g) and hypoglycosylation human tissues, consistent with the multi-organ pheno- of α-DG by both immunofluorescence and western blot- types of many individuals with the most severe forms of ting (Fig. 2). In contrast, α-DG in cultured fibroblasts was dystroglycanopathy [4]. indistinguishable from control fibroblasts in on-cell and in Mutations in DAG1 itself as well as 17 other genes WGA glycoprotein western blots (data not shown). This is have been reported in patients with dystroglycanopathy. not uncommon and has been reported in the case of other These include glycosyltransferases (POMT1, POMT2, genes involved in dystroglycanopathy [6, 7]. POMGNT1, POMGNT2, B3GALNT2, B3GNT1, LARGE, The patient had several subsequent acute infectious TMEM5), a kinase (POMK), five genes encoding en- illnesses with seizures and neurological regression. zymes necessary for dolichol-P-mannose (dol-P-man) Follow-up brain MRI at 15 months of age showed synthesis (DOLK, DPM1, DPM2, DPM3, and GMPPB), marked progressive volume loss (Fig. 1b). Glycosylation and three genes encoding proteins necessary for joining analysis of transferrin and ApoCIII proteins in serum by the α-DG-linked core glycan structure with the distal affinity chromatography-mass spectrometry (Mayo ligand-binding region of the structure via a ribitol Medical Laboratory) as well as by MALDI-TOF mass phosphate disaccharide (FKTN, FKRP, ISPD)[5]. To spectrometry (Emory Genetics Laboratory) was normal. date, no membrane trafficking proteins have been impli- In her last evaluation, at 3 years and 6 months of age, cated in dystroglycanopathies. she was areflexic with limited antigravity strength and In this study, we report clinical, histopathological, severe hypotonia. She fed exclusively via gastrostomy biochemical, and molecular genetic data on two families tube with no verbal communication. Seizures were well with CMD and hypoglycosylation of α-DG. Two genes, controlled on levetiracetam monotherapy. CK remained TRAPPC11 and GOSR2, that each have a role in mem- elevated with values as high as 19,000 U/L. She had mild brane trafficking in the biosynthetic pathway have been hepatomegaly and aminotransferases were still signifi- implicated as candidate dystroglycanopathy genes. They cantly elevated with 4:1 ALT to AST ratio, but there was represent the first membrane trafficking proteins impli- no coagulopathy or hyperbilirubinemia. She chronically cated in α-DG hypoglycosylation. Since TRAPPC11 required noninvasive positive pressure ventilation with mutations have been reported in a number of individuals sleep. After multiple hospital admissions for viral suffering from a muscular dystrophy, and these individ- respiratory infections, she underwent immunological uals also display membrane trafficking defects in cul- evaluation and was found to have impaired natural killer tured fibroblasts, this gene should be considered in the cell function on multiple repeated analyses. She did not diagnostic evaluation of patients with CMD. have peripheral neuropathy, cataracts, alacrima, achala- sia, renal disease, hearing loss, or cholestasis. Case presentation Exome trio sequencing showed compound heterozy- Family 1 gous rare variants in trans in TRAPPC11 (NM_021942): Subject 1 presented with status epilepticus in the setting c.851A>C (p.Q284P) and c.965+5G>T (intron 9 splice of a vomiting illness at 6 months of age. Magnetic reson- site disruptor). The p.Q284P missense mutation was ance imaging (MRI) of the brain showed bilateral multi- absent from the Exome Aggregation Consortium (ExAC) focal restricted diffusion of the cortex, the cerebral white database, and c.965+5G>T was present in 2/119,770 matter, and the pons (Fig. 1a). Her serum aminotransfer- alleles [8]. The latter mutation resulted in a transcript ases were elevated with alanine aminotransferase (ALT) that lacks exon 9 and the first 88 bases of exon 10 of ~ 1600 U/L and aspartate aminotransferase (AST) ~ (Fig. 3a) and is predicted to result in an in-frame dele- 400 U/L as well as a prolonged prothrombin time of tion of amino acids 278–351 (p.I278_Q351del). Cultured 20.7 s (normal range is 12–15 s), consistent with syn- fibroblasts had greatly reduced levels of TRAPPC11 thetic liver dysfunction. The approximately 4:1 ALT to (Fig. 3b) suggesting the p.Q284P protein and the pre- AST ratio was consistent over multiple measurements. dicted p.I278_Q351del protein are unstable. These fibro- Creatine kinase (CK) at initial presentation was 3500 U/ blasts showed a delay in the maturation of the marker Larson et al. Skeletal Muscle (2018) 8:17 Page 3 of 10 Fig. 1 Subjects 1 and 3 display brain, liver, and muscle abnormalities. a Diffusion-weighted (B1000) MRI showing restricted diffusion of the medial occipital cortex and underlying white matter at 6 months of age in subject 1 at the time of initial presentation. b Fluid-attenuated inversion recovery (FLAIR) MRI for subject 1 at 15 months notable for marked cerebral volume loss. c Short tau inversion recovery (STIR) shows symmetric high signal in the posterior compartments of the legs of subject 1 at 12 months of age. Subject 1 has microvesicular steatosis of the liver; light microscopy hematoxylin and eosin (d) and electron microscopy (e). Note the lipid accumulations marked by the arrows in e. f–h Muscle biopsies from control (f), subject 1 (g), and subject 3 (h) were stained with hematoxylin and eosin. Dystrophic features are present in subjects 1 and 3. The size bar denotes 50 μmin d and f–h. The size bar denotes 5 μmin e protein VSVG-GFP ts045 (Fig. 3c, d). Analysis of 9 months, she was areflexic. She had low muscle bulk live-cell trafficking revealed a delay in the release of and myopathic facial appearance and did not have anti- VSVG-GFP ts045 from the Golgi (Fig. 3e, f) as well as a gravity strength. She had fine nystagmus but otherwise delay in arrival of a Golgi marker (sialyl intact extraocular movements. MRI of the brain was transferase-SBP-GFP) from the endoplasmic reticulum normal at ages 2 and 4 years. She died due to respiratory (Fig. 3g, h). The delayed release of protein from the failure at age 5 years. Golgi is consistent with the initial findings reported by Subject 3 is the younger sister of subject 2. She was Bögershausen et al. in LGMD2S patients with noted to have muscle weakness and hypotonia at TRAPPC11 mutations [9], and the delayed arrival of 3 months of age. At 9 months, she had antigravity protein to the Golgi is consistent with the findings of strength only. Serum CK value was 1760 U/L. At Scrivens et al. [10]. 19 months, skeletal muscle biopsy was obtained showing an active dystrophic process (Fig. 1h) and hypoglycosyla- Family 2 tion of α-DG by both immunofluorescence and western Subject 2 presented for medical evaluation at age blotting (Fig. 2). In contrast, α-DG in cultured fibro- 6 months for hypotonia. She was found to have CK blasts was indistinguishable from control fibroblasts in values of up to ~ 5000 U/L. She developed absence on-cell and in WGA glycoprotein western blots (data seizures at age 2 years. She had steadily progressive not shown). Furthermore, the VSVG-GFP membrane muscle weakness. On examination at age 4 years and trafficking assay kinetics as well as arrival of the Golgi Larson et al. Skeletal Muscle (2018) 8:17 Page 4 of 10 Fig. 2 Subjects 1 and 2 display abnormalities in both α-dystroglycan staining and glycosylation. Control muscle or muscle taken from subject 1 (S1) and subject 3 (S3) were stained for alpha dystroglycan using VIA4-1 antibody (a)or β-DG (b). Note the reduced staining for α-DG but not β-DG in subjects 1 and 3. The size bar denotes 50 μm for all panels in a and b. c Western blot analysis of muscle tissue from control and subjects 1 and 3. Samples were probed with peptide-specific antibody AF6868 and the glycoepitope-specific antibody IIH6 as indicated. The location of α-DG and β-DG is indicated. Note that control shows a higher molecular size immunoreactive species for α-DG with both antibodies while S1 and S3 show a more heterogeneous species of much smaller molecular size, suggesting hypoglycosylation of the protein marker was indistinguishable from control fibroblasts Physical exam revealed findings similar to her sister. (Fig. 3c–f). Apart from her sister, there is no family history of At age 2.5 years, she developed a seizure disorder neuromuscular disease. The subject is now 6 years of characterized as focal seizures but later as both focal and age with medically refractory epilepsy and progressive generalized, which often became intractable and re- severe muscle weakness. Clinical exome trio sequencing quired hospitalization. Evaluation showed no evidence of was performed, and no relevant sequence variants were nystagmus and ocular range of motion was full. There initially reported. In a targeted sequencing panel, subject were no focal deficits and her cranial nerves were nor- 3 was found to have compound heterozygous rare vari- mal. She demonstrated severe weakness and muscular ants in GOSR2 (NM_001012511): c.430G>T (p.G144W) hypotonia. MRI of the brain showed diffuse volume loss and c.2T>G. Retrospective evaluation of GOSR2 in the resulting in ex vacuo ventriculomegaly. EEG at 2 years whole exome sequencing (WES) data confirmed that and 7 months of age showed runs of spike and wave both variants were present in subject 3 and were in discharges originating in the occipital lobe which were trans. Extensive re-evaluation of seizure and dystrogly- exacerbated by photic stimuli. Head circumference was canopathy loci in the WES failed to identify any other at the 30th centile, height at the 10th centile, and weight pathologic variants. The GOSR2 p.G144W missense below the 1st centile. variant is a previously reported disease-causing muta- At 3.5 years of age, she developed episodes of vomiting tion and is present in 5/121,408 alleles in the ExAC and apparent abdominal pain. This led to the detection database with no homozygous individuals. The second of elevated ALT of up to 700 U/L. An extensive evalu- variant (c.2T>G) is present in 1/18,808 alleles in the ation for infectious, anatomical, autoimmune, and meta- ExAC database [8]. The mutation is likely to result in bolic etiologies of liver disease was nondiagnostic. the use of an alternate start codon with elimination of Larson et al. Skeletal Muscle (2018) 8:17 Page 5 of 10 Fig. 3 (See legend on next page.) Larson et al. Skeletal Muscle (2018) 8:17 Page 6 of 10 (See figure on previous page.) Fig. 3 TRAPPC11 compound heterozygous mutations affect membrane trafficking in patient fibroblasts. a mRNA was collected from control and subject 1 (S1), converted to cDNA and amplified by PCR using oligonucleotides annealing to exons 8 and 11. The amplicons were sequenced and found to represent exons 8-9-10-11 (higher molecular size amplicon) and exons 8-part of 10-11 (lower molecular size amplicon). b Lysates from control and subject 1 (S1) fibroblasts were probed for TRAPPC11 and tubulin as a loading control. c Fibroblasts were infected with VSVG- GFP ts045, and the protein was arrested in the endoplasmic reticulum (ER) by shifting the cells to 40 °C. The protein was synchronously released from the ER upon downshifting the temperature to 32 °C, and the acquisition of Endoglycosidase H (EndoH) resistance was assayed at the times indicated. A representative western blot is displayed, and quantification of a minimum of three such blots is shown in d. e The same assay as in b was performed on live cells and the arrival and release of the GFP signal was quantified over time. Representative images from the movies are displayed in e, and quantification of the signal in the Golgi region is shown in f. To more accurately measure ER-to-Golgi trafficking, the RUSH assay [36] was performed using ST-SBP-GFP with the Ii hook (g). Images were acquired over time in live cells upon addition of biotin to initiate release of the protein from the ER. Quantification of the signal in the Golgi is displayed in h. Size bars in e and g denote 25 μm. Error bars represent SEM from a minimum of three replicates in d. N values for f and h are indicated in the figure 18 amino acids from the amino-terminus of the protein disease is a disorder of glycosylation, analysis of glycoepi- according to MutationTaster2 and is presumed to be topes of secreted proteins may not be a sensitive test for pathogenic as a result [11]. diagnostic purposes. The zebrafish model of TRAPPC11-related disease shows Discussion and conclusions generalized impairment of N-linked glycosylation as well as In this report, we show that mutations in two genes depletion of lipid-linked oligosaccharides (LLOs) [17]. The encoding proteins involved in membrane trafficking, inability to synthesize dolichol-P-mannose (dol-P-man), a TRAPPC11 and GOSR2, are associated with CMD and lipid-linked saccharide, is a known cause of dystroglycano- dystroglycanopathy. Biallelic mutations in TRAPPC11 pathy [7]. Expression of multiple glycosylation-related genes were initially reported as the etiology of LGMD2S in (including the known etiologies of dystroglycanopathy 2013 [9] and have since been associated with a variety of gmppb, dpm1, dpm2,and dpm3) showed significant com- multisystemic phenotypic findings including intellectual pensatory upregulation in the trappc11 fish [17]. Interest- disability, seizures, microcephaly, cerebral atrophy, ingly, TRAPPC11 siRNA knockdown in HeLa cells had a cataracts, alacrima, achalasia, hepatic steatosis, and specific inhibitory effect on glycosylation that was not cholestatic liver disease, in addition to muscular present with knockdown of other components of the dystrophy [9, 12–15]. Comparisons between subject 1 TRAPP complex. This led to the conclusion that and all published mutations in TRAPPC11 and associ- TRAPPC11 may have another function that is independent ated phenotypes are summarized in Table 1. Our study of its role in vesicle transport and led to speculation that now adds two new mutations with functional validation impaired LLO synthesis may be the most relevant function and categorizes TRAPPC11-related disease as a of TRAPPC11 in the process of protein glycosylation [17]. dystroglycanopathy. Finally, trappc11 zebrafish mutations were shown to lead to TRAPPC11 dysfunction may contribute to disease fatty liver via a pathological activation of the unfolded pathophysiology in several ways. Extensive functional protein response. This may be relevant to subject 1 as well studies of cultured fibroblasts were conducted by as the other reported individuals with hepatopathy and Bögershausen et al. [9]. They demonstrated that cells TRAPPC11-related disease [13]. Taken together, several had abnormally fragmented and diffuse Golgi; delayed mechanisms for the role of TRAPPC11 in muscular and traffic out of the Golgi and the proteins LAMP1 and hepatic phenotypes are known and can explain many LAMP2 were found to be abnormally glycosylated. clinical features of subject 1. TRAPP (transport protein particle) forms several related Human mutations in GOSR2 were first reported in multisubunit trafficking complexes (MTCs) that partici- 2011 in six individuals with the same homozygous pate in the tethering of vesicles to target membranes, missense mutation (c.430G>T) who had progressive including vesicles associated with the Golgi [10]. Since myoclonus epilepsy (PME), ataxia, scoliosis, and mildly the Golgi is the major site of protein glycosylation in the elevated serum CK (see Table 1 for a comparison cell [16], defects in Golgi morphology and traffic can between subjects 2 and 3 with all reported GOSR2 muta- result in protein glycosylation defects. Recently, abnor- tions) [18]. All individuals were areflexic in early child- mal glycosylation of serum transferrin was described in hood and were non-ambulatory by adolescence or early a patient with compound heterozygous mutations in adulthood. Muscle histology and EMG were normal. An TRAPPC11, consistent with a type 2 disorder of glyco- additional eleven individuals with similar clinical presen- sylation [12]. We were unable to detect abnormalities in tations and the same homozygous mutation were glycosylation of serum transferrin using two different com- reported in 2013 and 2014 [19, 20]. The maximum CK monly employed methods. Thus, while TRAPPC11-related value reported in any of the patients was 2467 U/L. Larson et al. Skeletal Muscle (2018) 8:17 Page 7 of 10 Table 1 Comparison of all known TRAPPC11 and GOSR2 mutations Genotype Number of Neurological phenotype Muscle phenotype Other features References cases TRAPPC11 3 motor delay in one individual, otherwise LGMD, CK up to ~ 2800 scoliosis, cataracts and esotropia Bogershausen et al. [9] c.2938G>A normal each in one individual c.2938G>A TRAPPC11 5 epilepsy, developmental delay, ataxia, myopathy, CK up to ~ 1200 short stature, exophoria in one individual Bogershausen et al. [9] c.1287+5G>A chorea, microcephaly, cerebral atrophy c.1287+5G>A TRAPPC11 4 developmental delay, cerebral atrophy, CMD, CK not reported, dystrophic scoliosis, achalasia, alacrima Koehler et al. [14] c.1893+3A>G medically refractory epilepsy appearance of biopsied muscle tissue c.1893+3A>G TRAPPC11 2 moderate intellectual disability, ambulatory, CMD, CK up to ~ 10,000; dystrophic cataracts, significantly elevated ALT, mildly Fee et al. [15] c.513_516delTTTG seizures, MRI with mild atrophy biopsied muscle, abnormal dystroglycan elevated AST, liver fibrosis c.2330A>C staining TRAPPC11 1 developmental delay, decreased white CMD, CK up to ~ 9000; abnormal signal hepatic steatosis, significantly elevated ALT, Liang et al. [13] c.2938G>A matter volume on MRI in posterior compartment leg muscles mildly elevated AST, cataracts c.661-1G>T on CT scan TRAPPC11 1 microcephaly, brain atrophy on MRI, presumed CMD, hypotonia, CK not retrognathia, cholestatic liver disease, Matalonga et al. [12] c.1141C>G sensorineural hearing loss, peripheral reported thrombocytopenia, nephropathy, osteopenia c.3310A>G neuropathy TRAPPC11 1 severe developmental delay, multifocal CMD, CK up to ~ 18,000; abnormal signal hepatic steatosis, significantly elevated ALT, This paper c.851A>C restricted diffusion on MRI; later cerebral in posterior compartment leg muscles on mildly elevated AST; retinopathy, impaired c.965+5G>T atrophy MRI scan; dystrophic appearance of biopsied NK cell function muscle; hypoglycosylation of α-dystroglycan GOSR2 17 “North Sea” progressive myoclonus epilepsy; CK up to ~ 2500 but normal in some; no scoliosis, pes cavus, syndactyly in some, Lomax et al. [19], Egmond c.430G>T childhood-onset ataxia, loss of ambulation specific abnormalities reported in muscle delayed puberty in some et al. [20], Corbett et al. [18] c.430G>T in early adulthood biopsies GOSR2 1 progressive myoclonus epilepsy, ataxia; MRI none reported none reported Praschberger et al. [37] c.430G>T with cerebral atrophy c.491_493delAGA GOSR2 2 medically refractory epilepsy; MRI with CMD, CK up to ~ 5000; dystrophic muscle no additional findings This paper c.430G>T cerebral atrophy biopsy with hypoglycosylation of c.2T>G α-dystroglycan; severe weakness and respiratory failure leading to death at 5 years in older sibling Larson et al. Skeletal Muscle (2018) 8:17 Page 8 of 10 There was no specific assessment of α-DG glycosylation addition of ribitol phosphate molecules to link the core in their muscle biopsies. Subjects 2 and 3 in our study and ligand-binding regions of the α-DG glycan structure have a much more severe phenotype. Since CMD [33–35]. Our study suggests that TRAPPC11 and represents the severe end of the clinical spectrum of GOSR2 are also involved in the trafficking and glycosyla- GOSR2-related disease and PME represents the milder tion of dystroglycan in the Golgi. This represents the end of the spectrum, the new c.2T>G mutation resulting first report of an association between these genes and in CMD reported in our study likely cause more severe α-DG hypoglycosylation. It remains to be seen if other perturbation of Golgi function than the common GOSR2 mutations associate with similar cellular and c.430G>T mutation. It remains unclear which aspect of clinical phenotypes. Given the number of individuals with Golgi function is affected since a membrane trafficking TRAPPC11 mutation-associated muscular dystrophy, it may defect in neither the VSVG-GFP marker protein nor a be prudent for this gene to now be considered in the diag- resident Golgi enzyme was detected. Future studies nostic evaluation of patients with dystroglycanopathy. should examine the trafficking of Golgi-localized Abbreviations glycosyl transferases that are responsible for α-DG ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; CK: Creatine processing. kinase; FLAIR: Fluid-attenuated inversion recovery; MRI: Magnetic resonance imaging; STIR: Short tau inversion recovery; α-DG: α-Dystroglycan GOSR2 encodes a Golgi Qb-SNARE (soluble N-ethyl- maleimide-sensitive factor attachment protein receptor) Acknowledgements protein. In the cell, GOSR2 localizes to the cis-Golgi and We are grateful to members of our laboratories for constructive comments on this report and for fruitful discussions. mediates docking and fusion of vesicles originating from the ER. There is precedent for Golgi dysfunction leading Funding to diseases manifesting with abnormal glycosylation and SAM and MOC are funded by the Iowa Wellstone Muscular Dystrophy Cooperative Research Center U54, NS053672. MS is funded by the Canadian multisystemic disease. Examples include the disease Institutes of Health Research and the Natural Sciences and Engineering caused by mutations in genes that encode the COG Research Council of Canada. RJS is funded by NCATS/NIH, UL1TR001082. (conserved oligomeric Golgi) complex, an MTC that Availability of data and materials localizes to the Golgi [21]. Additionally, an individual All data generated or analyzed during this study are included in this has been described with CMD due to homozygous mu- published article. tations in GOLGA2, a golgin protein that also impacts Authors’ contributions Golgi function [22]. The potential for a link between AAL designed the study and drafted the manuscript. PRB II designed the aberrant Golgi trafficking and dystroglycanopathy stems study and edited the manuscript. MPM performed the cell biological from an experiment employing a modified virus that membrane trafficking assays and edited the manuscript. CAP and RJS edited the manuscript. MOC grew the fibroblast cultures and performed western required normally glycosylated α-DG for cell entry. blots. JKL, AAS, and ADB developed the dystroglycanopathy sequencing Knockouts of known dystroglycanopathy genes in panel and performed genetic evaluation of subject 3. JMM evaluated the cultured fibroblasts resulted in impaired viral cell entry. sequencing data and provided a muscle biopsy for subject 3. KP performed the biochemical membrane trafficking assay. TFT analyzed the splicing defect Among the other knockouts shown to impair viral cell in subject 1. CAW evaluated the subject 3. MS designed the study and entry were those cells with mutations in several of the edited the manuscript. SAM designed the study, evaluated the muscle COG complex genes [23]. biopsy and cultured fibroblasts, coordinated the clinical groups, and edited the manuscript. All authors read and approved the final manuscript. Dystroglycanopathies result in muscular dystrophy due to dysfunctional linkage of the sarcolemma to the extra- Ethics approval and consent to participate cellular matrix. This linkage occurs via α-DG and relies All studies were completed according to local ethical approval of the institutional review boards. All individuals or their guardians gave written on the synthesis of a complex LARGE-glycan for normal informed consent before undergoing evaluation and testing, in agreement function [5]. Since the initial descriptions of dystroglyca- with the Declaration of Helsinki and approved by the ethical committees of the nopathy [1, 24–26], a variety of molecular mechanisms centers participating in this study, where biological samples were obtained. of the diseases have been discovered. Specific glycosyl- Consent for publication transferases such as POMT1/POMT2 are required to Consent for publication has been obtained by the participants or their legal construct the core glycan structure that is linked to guardians. α-DG [25, 26]. Mutations in DOLK, DPM1, DPM2, Competing interests DPM3, and GMPPB likely lead to a deficiency of The authors declare that they have no competing interests. dol-P-man (a lipid-linked monosaccharide) resulting in abnormal N-linked glycosylation as well as the O-linked Publisher’sNote mannosylation defect that results in dystroglycanopathy Springer Nature remains neutral with regard to jurisdictional claims in [6, 7, 27–31]. 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Skeletal MuscleSpringer Journals

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