Molecular diagnosis of hereditary inclusion body myopathy by linkage analysis and identification of a novel splice site mutation in GNE

Abstract

Background: Many myopathies share clinical features in common, and diagnosis often requires genetic testing. We ascertained a family in which five siblings presented with distal muscle weakness of unknown etiology.

Methods: We performed high-density genomewide linkage analysis and mutation screening of candidate genes to identify the genetic defect in the family. Preserved clinical biopsy material was reviewed to confirm the diagnosis, and reverse transcriptase PCR was used to determine the molecular effect of a splice site mutation.

Results: The linkage scan excluded the majority of known myopathy genes, but one linkage peak included the gene GNE, in which mutations cause autosomal recessive hereditary inclusion body myopathy type 2 (HIBM2). Muscle biopsy tissue from a patient showed myopathic features, including small basophilic fibers with vacuoles. Sequence analysis of GNE revealed affected individuals were compound heterozygous for a novel mutation in the 5' splice donor site of intron 10 (c.1816+5G>A) and a previously reported missense mutation (c.2086G>A, p.V696M), confirming the diagnosis as HIBM2. The splice site mutation correlated with exclusion of exon 10 from the transcript, which is predicted to produce an in-frame deletion (p.G545_D605del) of 61 amino acids in the kinase domain of the GNE protein. The father of the proband was heterozygous for the splice site mutation and exhibited mild distal weakness late in life.

Conclusions: Our study expands on the extensive allelic heterogeneity of HIBM2 and demonstrates the value of linkage analysis in resolving ambiguous clinical findings to achieve a molecular diagnosis.

Figure 1

Pedigree. Subjects with DNA samples available and tested are labeled with their identification number. Black-filled symbols represent individuals affected with HIBM2, with onset in their 20s or 30s. Tested patients were compound heterozygous with genotype NM_005476.5: c.[1816+5G>A]+[2086G>A]. The gray-filled symbol represents an individual presenting in his 70s with mild HIBM-like symptoms, with genotype NM_005476.5: c.[1816+5G>A]+[=]. Arrow indicates the proband.

Figure 2

Pathology characteristic of HIBM in biceps muscle biopsy from proband. A) Hematoxylin and eosin staining showed marked variation in fiber diameter, rounded fibers, mild endomysial fibrosis, slight fatty infiltration, and irregular vacuoles (arrows) containing granular material. Frankly necrotic fibers, basophilic regenerating fibers, and inflammatory infiltrates were not prominent. The disease process appeared focal, with fibers in some areas mildly affected, while other regions, including the one shown here, exhibited severe myopathic pathology. B) Modified Gomori trichrome staining highlighted small amounts of granular red material within and around the vacuoles (arrow), as well as endomysial fibrosis and small, angulated, atrophic fibers.

Figure 3

Correlation of GNE transcript length with c.1816+5G>A splice mutation genotype. RNA was extracted from muscle biopsy tissue for subject 1159-1 and from saliva for subjects 1159-2 and 1159-3. By capillary electrophoresis, RT-PCR produced a single band of the expected size in control samples and 1159-2, the mother of the proband, in whom the c.1816+5G>A mutation was absent. The proband (1159-1), and her father (1159-3), both of whom were heterozygous for the c.1816+5G>A mutation, showed the full length band and a shorter band that lacked exon 10.