Identification of an Alu element-mediated deletion in the promoter region of GNE in siblings with GNE myopathy

Abstract

Background: GNE myopathy is a rare genetic disease characterized by progressive muscle atrophy and weakness. It is caused by biallelic mutations in the GNE gene that encodes for the bifunctional enzyme, uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) 2-epimerase/N-acetylmannosamine (ManNAc) kinase. Typical characteristics of GNE myopathy include progressive myopathy, first involving anterior tibialis muscle and sparing the quadriceps, and rimmed vacuoles on muscle biopsy. Identifying biallelic mutations by sequencing of the GNE gene confirms the diagnosis of GNE myopathy. In a subset of patients, diagnostic confirmation is challenged by the identification of mutations in only one allele, suggesting mutations in deep intronic regions or regulatory regions.

Methods: We performed targeted sequencing and copy number variant (CNV) analysis of GNE in two siblings who clinically presented with GNE myopathy. Further molecular and biochemical studies were done to characterize the effect of a previously uncharacterized GNE mutation.

Results: We report two siblings of Indian descent with characteristic features of GNE myopathy, including progressive skeletal muscle weakness initially involving the anterior tibialis, and rimmed vacuoles on muscle biopsy, in which a heterozygous mutation, p.Val727Met, was identified in both affected siblings, but no other deleterious variants in either coding region or exon-intron boundaries of the gene. Subsequent insertion/deletion analysis identified a novel 11.3-kb deletion (Chr9 [GRCh37]: g.36257583_36268910del) encompassing the GNE promoter region, with breakpoints residing in Alu repeats. Gene expression analysis revealed reduced GNE mRNA and protein levels, confirming decreased expression of the deleted allele harboring the deletion.

Conclusions: We have identified GNE as one of the genes susceptible to Alu-mediated recombination. Our findings suggest that the deletion may encompass the promoter or another region necessary for GNE expression. In patients with typical manifestations of GNE myopathy and a single GNE variant identified, copy number variant (CNV) analysis may be useful in arriving at the diagnosis.

Keywords: Alu‐SINE repeat; GNE isoforms; GNE myopathy; array‐CGH; copy number variant; genomic rearrangement; precision medicine; sialic acid.

Figure 1

Muscle imaging and pathology. (A) T₁‐weighted MRI of the thigh (upper panel) and lower leg (lower panel). Atrophy of hamstrings and lower leg muscles (Patient 1) and the proximal anterior tibialis (Patient 2) gave rise to fatty infiltration, apparent as white on the MRI. (B) Muscle biopsies of biceps brachii and lower extremity muscles (gastrocnemius medialis in Patient 1, anterior tibialis in Patient 2, quadriceps femoris in Normal) show characteristic findings of GNE myopathy, including rimmed vacuoles (arrows), fatty and fibrous tissue replacement (double arrows), marked variation in fiber size, and central nucleation (arrowhead). Note that the biceps muscle of Patient 2 appears normal, except for a mild variation in fiber size. Scale bar = 50 microns.

Figure 2

GNE mutation analysis. (A) Sanger sequencing of GNE exon 13 confirmed the heterozygous GNE mutation [NM_001128227.2:c.2179G>A;p.Val727Met] in both siblings (Patient 1 displayed). (B) Enlarged region of chromosome 9 (GRCh37) aCGH analysis that showed heterozygosity of a large (>10 kb) region in both patients. Primer locations for deletion analysis are indicated (not to scale, note that GNE gene is transcribed on reverse strand). (C) The size and breakpoints of the deletion were established by PCR analysis across the deletion; expected fragment sizes for each primer set are indicated. (D) Sanger sequencing across the deletion (Primer 1R; reverse sequence shown) determined the exact breakpoints and deletion size as: Chr9(GRCh37):g.36257583_36268910del (del 11,328‐bp).

Figure 3

GNE gene and protein expression. (A) Display of the 5′ terminal region of the five GNE splice variants listed in GenBank. Exons represented by dotted boxes are absent in respective transcripts. The 11.3 kb deletion breakpoints (vertical dotted lines) within Alu‐repeat regions (black boxes) and the start codon (ATG) position (arrow) of each isoform is shown. The deletion is located deeply intronic for two isoforms (hGNE2 and hGNE3) and in the 5′ UTR and promoter region of others (hGNE1,hGNE4, and hGNE5). (B) Blood GNE mRNA levels normalized with POLR2A. Patients 1 and 2, harboring the 11.3 kb GNE deletion in combination with a missense, have reduced GNE expression, as compared to other GNE myopathy patients whose biallelic missense mutations in GNE are mentioned. *P < 0.05. (C) Measurement of GNE protein expression in lymphoblastoid cell lines from control and Patients 1 and 2. Expression levels were normalized with ACTB, and expressed as a ratio with control (lower bar graph). *P < 0.05.