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

Abstract

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.

Keywords: GNE myopathy (HIBM); aCGH; molecular diagnostics; myositis; next generation sequencing; transcriptome sequencing (RNA-seq).

FIGURE 1.

Muscle biopsy showing central and sub-sarcolemmal vacuoles in hemotoxylin and eosin (H&E) (top panel left), which demonstrates red “rimming” with modified Gomori trichrome (top center panel). An example of inflammation is seen in the top right panel (circled). Bottom panel: Acid phosphatase (left) and NADH (center) showing positive material within the vacuoles and vacuoles are stained positive for ubiquitin (black arrows) (right). Scale bar = 50 μm. Additionally, the connective tissue was mildly increased. Atrophic fibers were round and pyknotic nuclear clumps were not seen, and the biopsy showed a moderate number of fibers with internalized nuclei. Regenerating fibers were not seen. The following stains were normal: cytochrome oxidase (COX), myosin ATPase (normal distribution of fiber types), Oil red O, periodic acid–Schiff (PAS), phosphorylase, Congo red. Neither muscle fiber-type grouping, nor type specific atrophy was seen.

FIGURE 2.

(A) Integrated Genomics Viewer (IGV) pile up of RNA-sequencing showing monoallelic expression of GNE gene with only the allele harboring c.2179C>T:G>A (p.V727M) missense “likely pathogenic” variant expressed. The red arrow indicates the position of the V727M variant in exon 13 of the GNE gene. Sanger sequencing confirmation was performed on cDNA showing monoallelic expression as shown below. (B) aCGH signal showing a deletion upstream of the GNE gene with genomic breakpoints at nucleotide positions g.36,259,402 and 36,266,483 was detected in this individual (SCV000599234). This deletion is 7.08 kb in size and encompasses the untranslated exon 2 of the hGNE2 transcript but upstream of the hGNE1 transcript of the GNE gene. (C,D) Exome sequencing and later aCGH of trios reveal that monoallelic expression was due to expression of only the paternal allele of GNE in the proband.

FIGURE 3.

(A) Approximately 50% lower-expression (P < 0.05) of GNE in GNE myopathy patient muscle compared with that in 6 control normal muscle biopsies. (B) IIH6-antibody against glycosylated-α-DG shows hypoglycosylation (lighter signal, broader smear) of α-DG in patient muscle compared with control. AF6868 against core α-DG, β-DG shows different α-DG smearing patterns in patient muscle compared with control. Two predominant staining areas are 150 kDa and 100 kDa in patient sample. Roughly same β-DG-fragment expression at ~43 kDa is seen in both patient and control muscles. (C) HIBM-causing point mutations (blue, purple sticks) mapped onto GNE-N-acetylmannosamine-kinase-domain (PDB ID: 2YHY); N-acetylmannosamine-kinase-dimer backbone shown as cartoon. Protomers are colored white and cyan, associated divalent ions as spheres (chloride ions: green, zinc ion: lavender). Active site bound ADP and N-acetylmannosamine are shown as sticks on the right hand side GNE protomer. HIBM-causing residues are mapped on the left hand side GNE protomer. The V727M (V696M) mutation (purple sticks), other HIBM mutations (blue sticks). (D) Valine-727, and other HIBM mutations, are shown on both dimer subunits. (E) The Valine-727 sidechain is replaced by a Methionine residue on both dimer subunits and colored in Corey, Pauling, Koltun scheme (oxygen in red and sulfur in yellow). (F) The rotamer is changed upon replacing V727 (green sticks) by a methionine (red sticks) in MD simulations.