Clinical utility of RNA sequencing to resolve unusual GNE myopathy with a novel promoter deletion.

INTRODUCTION: UDP N-acetylglucosamine2-epimerase/N-acetylmannosamine-kinase (GNE) gene mutations can cause mostly autosomal-recessive myopathy with juvenile-onset known as hereditary inclusion-body myopathy (HIBM). METHODS: We describe a family of a patient showing an unusual HIBM with both vacuolar myopathy and myositis without quadriceps-sparing, hindering diagnosis. We show how genetic testing with functional assays, clinical transcriptome sequencing (RNA-seq) in particular, helped facilitate both the diagnosis and a better understanding of the genotype-phenotype relationship. RESULTS: We identified a novel 7.08 kb pathogenic deletion upstream of GNE using array comparative genomic hybridization (aCGH) and a common Val727Met variant. Using RNA-seq, we found only monoallelic (Val727Met-allele) expression, leading to ~50% GNE reduction in muscle. Importantly, α-dystroglycan is hypoglycosylated in the patient muscle, suggesting HIBM could be a "dystroglycanopathy." CONCLUSIONS: Our study shows the importance of considering aCGH for GNE-myopathies, and the potential of RNA-seq for faster, definitive molecular diagnosis of unusual myopathies. Muscle Nerve, 2019.

Structural and biophysical analyses of the skeletal dihydropyridine receptor ß subunit ß1a reveal critical roles of domain interactions for stability.

Excitation-contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel dihydropyridine receptor (DHPR) and the ryanodine receptor Ca2+ release channel. Although the exact molecular mechanism that initiates skeletal EC coupling is unresolved, it is clear that both the α1 and ß subunits of DHPR are essential for this process. Here, we employed a series of techniques, including size-exclusion chromatography-multi-angle light scattering, differential scanning fluorimetry, and isothermal calorimetry, to characterize various biophysical properties of the skeletal DHPR ß subunit ß1a Removal of the intrinsically disordered N and C termini and the hook region of ß1a prevented oligomerization, allowing for its structural determination by X-ray crystallography. The structure had a topology similar to that of previously determined ß isoforms, which consist of SH3 and guanylate kinase domains. However, transition melting temperatures derived from the differential scanning fluorimetry experiments indicated a significant difference in stability of ∼2-3 °C between the ß1a and ß2a constructs, and the addition of the DHPR α1s I-II loop (α-interaction domain) peptide stabilized both ß isoforms by ∼6-8 °C. Similar to other ß isoforms, ß1a bound with nanomolar affinity to the α-interaction domain, but binding affinities were influenced by amino acid substitutions in the adjacent SH3 domain. These results suggest that intramolecular interactions between the SH3 and guanylate kinase domains play a role in the stability of ß1a while also providing a conduit for allosteric signaling events.

Synthesis of peptides and glycopeptides with polyproline II helical topology as potential antifreeze molecules.

A library of peptides and glycopeptides containing (4R)-hydroxy-L-proline (Hyp) residues were designed with a view to providing stable polyproline II (PPII) helical molecules with antifreeze activity. A library of dodecapeptides containing contiguous Hyp residues or an Ala-Hyp-Ala tripeptide repeat sequence were synthesized with and without α-O-linked N-acetylgalactosamine and α-O-linked galactose-ß-(1→3)-N-acetylgalactosamine appended to the peptide backbone. All (glyco)peptides possessed PPII helical secondary structure with some showing significant thermal stability. The majority of the (glyco)peptides did not exhibit thermal hysteresis (TH) activity and were not capable of modifying the morphology of ice crystals. However, an unglycosylated Ala-Hyp-Ala repeat peptide did show significant TH and ice crystal re-shaping activity suggesting that it was capable of binding to the surface of ice. All (glyco)peptides synthesized displayed some ice recrystallization inhibition (IRI) activity with unglycosylated peptides containing the Ala-Hyp-Ala motif exhibiting the most potent inhibitory activity. Interestingly, although glycosylation is critical to the activity of native antifreeze glycoproteins (AFGPs) that possess an Ala-Thr-Ala tripeptide repeat, this same structural modification is detrimental to the antifreeze activity of the Ala-Hyp-Ala repeat peptides studied here.

N-terminal domain on dystroglycan enables LARGE1 to extend matriglycan on α-dystroglycan and prevents muscular dystrophy.

Dystroglycan (DG) requires extensive post-translational processing and O-glycosylation to function as a receptor for extracellular matrix (ECM) proteins containing laminin-G (LG) domains. Matriglycan is an elongated polysaccharide of alternating xylose (Xyl) and glucuronic acid (GlcA) that binds with high affinity to ECM proteins with LG domains and is uniquely synthesized on α-dystroglycan (α-DG) by like-acetylglucosaminyltransferase-1 (LARGE1). Defects in the post-translational processing or O-glycosylation of α-DG that result in a shorter form of matriglycan reduce the size of α-DG and decrease laminin binding, leading to various forms of muscular dystrophy. Previously, we demonstrated that protein O-mannose kinase (POMK) is required for LARGE1 to generate full-length matriglycan on α-DG (~150-250 kDa) (Walimbe et al., 2020). Here, we show that LARGE1 can only synthesize a short, non-elongated form of matriglycan in mouse skeletal muscle that lacks the DG N-terminus (α-DGN), resulting in an ~100-125 kDa α-DG. This smaller form of α-DG binds laminin and maintains specific force but does not prevent muscle pathophysiology, including reduced force production after eccentric contractions (ECs) or abnormalities in the neuromuscular junctions. Collectively, our study demonstrates that α-DGN, like POMK, is required for LARGE1 to extend matriglycan to its full mature length on α-DG and thus prevent muscle pathophysiology.

Deep Mutational Scanning in Disease-related Genes with Saturation Mutagenesis-Reinforced Functional Assays (SMuRF).

Interpretation of disease-causing genetic variants remains a challenge in human genetics. Current costs and complexity of deep mutational scanning methods hamper crowd-sourcing approaches toward genome-wide resolution of variants in disease-related genes. Our framework, Saturation Mutagenesis-Reinforced Functional assays (SMuRF), addresses these issues by offering simple and cost-effective saturation mutagenesis, as well as streamlining functional assays to enhance the interpretation of unresolved variants. Applying SMuRF to neuromuscular disease genes FKRP and LARGE1, we generated functional scores for over 99.8% of all possible coding single nucleotide variants and resolved 310 clinically reported variants of uncertain significance with high confidence, enhancing clinical variant interpretation in dystroglycanopathies. SMuRF also demonstrates utility in predicting disease severity, resolving critical structural regions, and providing training datasets for the development of computational predictors. Our approach opens new directions for enabling variant-to-function insights for disease genes in a manner that is broadly useful for crowd-sourcing implementation across standard research laboratories.

Identification of a short, single site matriglycan that maintains neuromuscular function in the mouse.

Matriglycan (-1,3-ß-glucuronic acid-1,3-α-xylose-) is a polysaccharide that is synthesized on α-dystroglycan, where it functions as a high-affinity glycan receptor for extracellular proteins, such as laminin, perlecan and agrin, thus anchoring the plasma membrane to the extracellular matrix. This biological activity is closely associated with the size of matriglycan. Using high-resolution mass spectrometry and site-specific mutant mice, we show for the first time that matriglycan on the T317/T319 and T379 sites of α-dystroglycan are not identical. T379-linked matriglycan is shorter than the previously characterized T317/T319-linked matriglycan, although it maintains its laminin binding capacity. Transgenic mice with only the shorter T379-linked matriglycan exhibited mild embryonic lethality, but those that survived were healthy. The shorter T379-linked matriglycan exists in multiple tissues and maintains neuromuscular function in adult mice. In addition, the genetic transfer of α-dystroglycan carrying just the short matriglycan restored grip strength and protected skeletal muscle from eccentric contraction-induced damage in muscle-specific dystroglycan knock-out mice. Due to the effects that matriglycan imparts on the extracellular proteome and its ability to modulate cell-matrix interactions, our work suggests that differential regulation of matriglycan length in various tissues optimizes the extracellular environment for unique cell types.

Lassa Fever Virus Binds Matriglycan-A Polymer of Alternating Xylose and Glucuronate-On α-Dystroglycan.

Lassa fever virus (LASV) can cause life-threatening hemorrhagic fevers for which there are currently no vaccines or targeted treatments. The late Prof. Stefan Kunz, along with others, showed that the high-affinity host receptor for LASV, and other Old World and clade-C New World mammarenaviruses, is matriglycan-a linear repeating disaccharide of alternating xylose and glucuronic acid that is polymerized uniquely on α-dystroglycan by like-acetylglucosaminyltransferase-1 (LARGE1). Although α-dystroglycan is ubiquitously expressed, LASV preferentially infects vascular endothelia and professional phagocytic cells, which suggests that viral entry requires additional cell-specific factors. In this review, we highlight the work of Stefan Kunz detailing the molecular mechanism of LASV binding and discuss the requirements of receptors, such as tyrosine kinases, for internalization through apoptotic mimicry.

POMK regulates dystroglycan function via LARGE1-mediated elongation of matriglycan.

Matriglycan [-GlcA-ß1,3-Xyl-α1,3-]n serves as a scaffold in many tissues for extracellular matrix proteins containing laminin-G domains including laminin, agrin, and perlecan. Like-acetyl-glucosaminyltransferase 1 (LARGE1) synthesizes and extends matriglycan on α-dystroglycan (α-DG) during skeletal muscle differentiation and regeneration; however, the mechanisms which regulate matriglycan elongation are unknown. Here, we show that Protein O-Mannose Kinase (POMK), which phosphorylates mannose of core M3 (GalNAc-ß1,3-GlcNAc-ß1,4-Man) preceding matriglycan synthesis, is required for LARGE1-mediated generation of full-length matriglycan on α-DG (~150 kDa). In the absence of Pomk gene expression in mouse skeletal muscle, LARGE1 synthesizes a very short matriglycan resulting in a ~ 90 kDa α-DG which binds laminin but cannot prevent eccentric contraction-induced force loss or muscle pathology. Solution NMR spectroscopy studies demonstrate that LARGE1 directly interacts with core M3 and binds preferentially to the phosphorylated form. Collectively, our study demonstrates that phosphorylation of core M3 by POMK enables LARGE1 to elongate matriglycan on α-DG, thereby preventing muscular dystrophy.

The structure of an LIM-only protein 4 (LMO4) and Deformed epidermal autoregulatory factor-1 (DEAF1) complex reveals a common mode of binding to LMO4.

LIM-domain only protein 4 (LMO4) is a widely expressed protein with important roles in embryonic development and breast cancer. It has been reported to bind many partners, including the transcription factor Deformed epidermal autoregulatory factor-1 (DEAF1), with which LMO4 shares many biological parallels. We used yeast two-hybrid assays to show that DEAF1 binds both LIM domains of LMO4 and that DEAF1 binds the same face on LMO4 as two other LMO4-binding partners, namely LIM domain binding protein 1 (LDB1) and C-terminal binding protein interacting protein (CtIP/RBBP8). Mutagenic screening analysed by the same method, indicates that the key residues in the interaction lie in LMO4LIM2 and the N-terminal half of the LMO4-binding domain in DEAF1. We generated a stable LMO4LIM2-DEAF1 complex and determined the solution structure of that complex. Although the LMO4-binding domain from DEAF1 is intrinsically disordered, it becomes structured on binding. The structure confirms that LDB1, CtIP and DEAF1 all bind to the same face on LMO4. LMO4 appears to form a hub in protein-protein interaction networks, linking numerous pathways within cells. Competitive binding for LMO4 therefore most likely provides a level of regulation between those different pathways.

Backbone and side-chain assignments of a tethered complex between LMO4 and DEAF-1.

The transcriptional regulator LMO4 and the transcription factor DEAF-1 are both essential for brain and skeletal development. They are also implicated in human breast cancers; overexpression of LMO4 is an indicator of poor prognosis, and overexpression of DEAF-1 promotes epithelial breast cell proliferation. We have generated a stable LMO4-DEAF-1 complex comprising the C-terminal LIM domain of LMO4 and an intrinsically disordered LMO4-interaction domain from DEAF-1 tethered by a glycine/serine linker. Here we report the (1)H, (15)N and (13)C assignments of this construct. Analysis of the assignments indicates the presence of structure in the DEAF-1 part of the complex supporting the presence of a physical interaction between the two proteins.

LIM-domain-only proteins in cancer.

LIM-domain proteins are a large family of proteins that are emerging as key molecules in a wide variety of human cancers. In particular, all members of the human LIM-domain-only (LMO) proteins, LMO1-4, which are required for many developmental processes, are implicated in the onset or the progression of several cancers, including T cell leukaemia, breast cancer and neuroblastoma. These small proteins contain two protein-interacting LIM domains but little additional sequence, and they seem to function by nucleating the formation of new transcriptional complexes and/or by disrupting existing transcriptional complexes to modulate gene expression programmes. Through these activities, the LMO proteins have important cellular roles in processes that are relevant to cancer such as self-renewal, cell cycle regulation and metastasis. These functions highlight the therapeutic potential of targeting these proteins in cancer.

Contribution of DEAF1 structural domains to the interaction with the breast cancer oncogene LMO4.

The proteins LMO4 and DEAF1 contribute to the proliferation of mammary epithelial cells. During breast cancer LMO4 is upregulated, affecting its interaction with other protein partners. This may set cells on a path to tumour formation. LMO4 and DEAF1 interact, but it is unknown how they cooperate to regulate cell proliferation. In this study, we identify a specific LMO4-binding domain in DEAF1. This domain contains an unstructured region that directly contacts LMO4, and a coiled coil that contains the DEAF1 nuclear export signal (NES). The coiled coil region can form tetramers and has the typical properties of a coiled coil domain. Using a simple cell-based assay, we show that LMO4 modulates the activity of the DEAF NES, causing nuclear accumulation of a construct containing the LMO4-interaction region of DEAF1.