L-2-hydroxyglutaric aciduria (L-2-HGA) is a neurometabolic disorder producing neurological deficits in human patients which include psychomotor retardation, progressive cerebellar dysfunction, learning disability and seizures.1,2 L-2-HGA is characterised by elevated levels of L-2-hydroxyglutaric acid (L-2-HG) in a variety of tissues and fluids, including CSF, plasma and urine. Mutations within the gene L2HGDH (Entrez Gene ID 79944) encoding L-2-hydroxyglutaric dehydrogenase have recently been shown to cause L-2-HGA in human patients. L-2-HGA has been identified within the Staffordshire bull terrier (SBT) and West Highland white terrier (WHWT), with affected dogs presenting between 6-months and 1-year of age (but up to 7-years) with ataxia, muscular stiffness at exercise or excitement, altered behaviour or epileptic seizures. In the dog, as in humans, L-2-HGA appears to be inherited as an autosomal recessive condition. A genetic screening test is available in the dog, allowing eradication of canine L-2-HGA.
L-2-HGA was first recognised in 1980 and has the biochemical hallmark of elevated levels of L-2-hydroxyglutaric acid (L-2-HG) in a variety of tissues and fluids, including cerebrospinal fluid, plasma and urine.3 L-2-hydroxyglutarate is converted into 2-oxoglutarate (a widely distributed compound formed through deamination of glutamate and formed within the Krebs cycle) by L-2-hydroxglutarate dehydrogenase, a FAD-dependent dehydrogenase linked to mitochondrial membranes. Three separate studies have recently identified mutations responsible for the condition in humans within the gene L2HGD (Entrez Gene ID 79944; previously known as C14orf160 [chromosome 14 open reading frame 160], duranin and FLJ12618) on chromosome 14q22.1,4,8,9 L2HGDH has been shown to encode L-2-HG dehydrogenase, with the demonstrated mutations within L2HGDH in L-2-HGA patients resulting in complete loss of L-2-HG dehydrogenase function and defective L-2-HG processing in an in vitro model.5,9 In the dog the mutation has also been demonstrated in the gene L2HGD, making it a true homologue of human L-2-HGA.
Urinary Organic Acid Analysis
The biological hallmark of L-2-HGA is the finding of elevated levels of L-2-hydroxyglutatic acid (HG) in a variety of tissues and fluids, including urine. Organic acid profiles by gas chromatography-mass spectrometry represent one of the more sophisticated screening methods currently available as a large number of metabolic disorders may be detected either directly or indirectly by this technique, including 2-HG. Gas chromatography-mass spectrometry demonstrates dramatic elevation of 2-HG in the urine of all affected dogs, but not in unaffected dogs or dogs carrying one abnormal copy of the gene. Urine can be stored frozen (collected with no preservative added) until analysis. Extraction of urinary organic acids is performed by specialised laboratory according to their individual technique, but the technique used in our laboratory is a modification of the protocol described by Tanaka and others.10 An aliquot of urine is made up relative to creatinine, to which an internal standard is added to allow quantification of the urinary organic acids. Once separated and derivatised, the organic acids are separated by gas chromatography and identified by mass spectrometry. Accumulation of 2-HA has been reported in the D- and L- isoforms in man, resulting in different disease phenotypes. Only L-2-HGA has been reported in dogs and the identification of 2-HG accumulation is therefore extremely suggestive of L-2-HGA, however differential diagnosis between L-2-H and D-2-HG and quantification of L-2-HG and D-2-HG can be performed by stable-isotope-dilution gas chromatography-mass spectrometry.11
Clinical Features in Affected Dogs
L-2-HGA in Staffordshire bull terrier and the West Highland white terrier affects both male and female dogs. The initial clinical signs are first evident between 6 months and one year of age (but in exceptional cases dogs may be up to 7 years of age). Puppies that are affected by the disease, but which were identified through a urine screening programme initially demonstrate no detectable clinical signs, despite demonstrable urine accumulation of L-2-HG. In dogs that do demonstrate clinical signs there is spectrum of clinical features and severity, however all affected dogs present with neurological signs, including one or more clinical signs of epileptic seizures, ataxia, hypermetria, tremors, muscular stiffness at exercise or excitement and altered behaviour (from mild behavioural changes to loss of obedience and loss of house training). There is currently no treatment available for this disease. Affected animals can be placed on medication to help control the clinical symptoms, most typically on phenobarbitone medication to control the epileptic seizures, but this does not address the underlying disease. Because of the absence of an effective treatment and the underlying genetic cause of this disease it is therefore important to focus on eliminating the disease from the population. The majority of dogs demonstrate gradual progression over a number of years, with many dogs having a reasonable quality of life for some years. However, in many cases euthanasia is requested, particularly if the gait deteriorates.
Magnetic Resonance Imaging (MRI) Features
Magnetic resonance (MR) imaging features in all affected dogs demonstrate a highly conserved pattern of bilaterally symmetrical regions of hyperintensity on T2W and FLAIR images similar to that observed in human patients.1,12-16 Within the cerebral cortex there is evidence of white matter hyperintensity, with sparing of the central region of the internal capsule and the corpus callosum and a zone of marked hyperintensity at the level of the grey-white matter interface. Consistent hyperintensity is evident in the caudal (inferior) colliculi, dorsomedian tegmentum, cerebellar nuclei (including the dentate nuclei) and thalamus. The basal nuclei, including the putamen and globus pallidus, are variably affected. On T1W images the parenchyma demonstrating hyperintensity on T2W images appears mildly hypointense. No abnormal contrast enhancement is evident.
Microsatellite homozygosity mapping has demonstrated that a mutation within L2HGDH is responsible for L-2-HGA in both the Staffordshire bull terrier and the West Highland white terrier. Sequencing of canine L2HGDH demonstrates a two base pair substitution in exon 10 of affected Staffordshire bull terriers and the introduction of a premature stop codon in exon 8 of affected West Highland white terriers, significantly altering the encoded amino acid sequence.17,18 If the sequencing data in the Staffordshire bull terrier is compared there are actually two mutations within the gene, i) two single nucleotide substitutions separated by a single invariant T nucleotide in exon 10 (c.[1297T>C; 1299c>t]; p.[Leu433Pro; His434Tyr]) and ii) a single nucleotide substitution in intron 8_9 (c.[1064+8C>T]). All nucleotide numbering is in accordance with the current full Ensembl gene build for Canis familiaris (CanFam 1.0: http://www.ensembl.org/Canis_familiaris/). Sequencing of both mutations demonstrated that all affected Staffordshire bull terriers were homozygous and all carrier dogs were heterozygous for the exon 10 double amino acid substitution. The mutation was not present in any of the normal related dogs (determined by homozygosity mapping data to be homozygous for the wild type haplotype), nor in a control population of unrelated, clinically normal Staffordshire bull terriers. The mutation causes the substitution of two amino acids from leucine and histidine to proline and tyrosine respectively. The presence of an identical mutation in all affected dogs suggests a common founder effect within the Staffordshire bull terrier breed. The mutation within the 5' flanking intron region of exon 8 did not modify an encoded amino acid and was homozygous in some of the normal carriers, indicating that this is likely to be a polymorphism with no pathological significance, however this mutation may potentially provide an explanation for the clinical heterogeneity seen in the disease in the Staffordshire bull terrier. These findings indicate that L-2-HGA is a recessive condition in the Staffordshire bull terrier and West Highland white terrier and that a mutation within the canine homologue of L2HGDH is responsible for L-2-HGA in affected animals.
Five generation pedigree analysis from the Staffordshire bull terrier, with analysis of additional extension pedigrees supplied by the Kennel Club, revealed that all affected Staffordshire bull terriers with pedigree information can be traced back to a common ancestry, supportive of a common founder effect. Due to a lack of pedigree information the same analysis has not been performed in the West Highland white terrier.
Dogs affected by L-2-HGA initially may not demonstrate clinical signs, but will invariably demonstrate evidence of diffuse CNS involvement, with a combination of forebrain and cerebellar signs. All affected dogs demonstrate dramatic accumulation of L-2-HG in urine and characteristic brain MRI changes. The disease is recessive and carrier dogs do not demonstrate clinical signs or accumulation of L-2-HG. A mutation within L2HGDH is responsible for canine L-2-HGA., with sequencing analysis demonstrating a two base pair substitution in exon 10 of affected SBTs and the introduction of a premature stop codon in exon 8 of affected WHWTs, significantly altering the encoded amino acid sequence. A genetic screening test has been developed in the Staffordshire bull terrier, allowing eradication of canine L-2-HGA.
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