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

Boyden SE, Duncan AR, Estrella EA, Lidov HG, Mahoney LJ, Katz JS, Kunkel LM, Kang PB - BMC Med. Genet. (2011)

Bottom Line: 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 father of the proband was heterozygous for the splice site mutation and exhibited mild distal weakness late in life.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Genetics, Children's Hospital Boston, Boston, MA 02115, USA.

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.

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Related in: MedlinePlus

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.
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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.

Mentions: An RT-PCR amplicon spanning exons 7 to 12 of the GNE transcript showed two products in the proband and her father, both of whom carried the c.1816+5G>A splice site mutation, whereas the proband's mother and control samples showed only the expected full-length band (Figure 3). Both bands were individually purified and sequenced from the proband and her father, and in both subjects the larger product was confirmed to represent the full-length amplicon, while the smaller product lacked exon 10. Skipping of exon 10, comprised of 183 base pairs, at the mRNA level would result in an in-frame deletion (p.G545_D605del) of 61 amino acids. Both the p.G545_D605del and p.V696M mutations are in the kinase domain of the GNE protein; mutations throughout the gene, in both the epimerase and kinase domains, can be pathogenic for HIBM2 [5].


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

Boyden SE, Duncan AR, Estrella EA, Lidov HG, Mahoney LJ, Katz JS, Kunkel LM, Kang PB - BMC Med. Genet. (2011)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141630&req=5

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.
Mentions: An RT-PCR amplicon spanning exons 7 to 12 of the GNE transcript showed two products in the proband and her father, both of whom carried the c.1816+5G>A splice site mutation, whereas the proband's mother and control samples showed only the expected full-length band (Figure 3). Both bands were individually purified and sequenced from the proband and her father, and in both subjects the larger product was confirmed to represent the full-length amplicon, while the smaller product lacked exon 10. Skipping of exon 10, comprised of 183 base pairs, at the mRNA level would result in an in-frame deletion (p.G545_D605del) of 61 amino acids. Both the p.G545_D605del and p.V696M mutations are in the kinase domain of the GNE protein; mutations throughout the gene, in both the epimerase and kinase domains, can be pathogenic for HIBM2 [5].

Bottom Line: 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 father of the proband was heterozygous for the splice site mutation and exhibited mild distal weakness late in life.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Genetics, Children's Hospital Boston, Boston, MA 02115, USA.

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.

Show MeSH
Related in: MedlinePlus