L-2-hydroxyglutaric Aciduria in Two Female Yorkshire Terriers

Introduction

L-2-hydroxyglutaric aciduria (L2HGA) in dogs is an autosomal recessive inborn error of intermediary metabolism showing a variety of progressive neurologic signs, including seizures, dementia, head tremors, muscle stiffness, and cerebellar ataxia (i.e., a wide-based stance, truncal sway, loss of balance, and dysmetric gait). L2HGA has been previously identified in Staffordshire bull terriers and in one West Highland white terrier.^1,2 The L2HGDH gene encodes for the mitochondrial membrane enzyme called L-2-hydroxyglutarate dehydrogenase. The L2HGDH gene is active in cells throughout the body, particularly in the brain, muscles, and testes. A mutation in the canine homolog gene L2HGDH on chromosome 8 has been shown to cause L2HGA in Staffordshire bull terriers.³

In this report, two cases of L2HGA in a third breed, the Yorkshire terrier, are described. Clinical, laboratory, and MRI features characteristic of the disease are detailed.

Case Report

Case 1

A 7 mo old female Yorkshire terrier weighing 2.0 kg was presented to her primary veterinarian with a 3 mo history of generalized tonic-clonic seizures and periodic obtundation. The owner had initially noted intermittent head pressing with episodic ataxia. Over the ensuing 3 mo, the owner noted an increased frequency and severity of the seizures. The dog had no travel history or known exposure to toxins, was fed a commercial meat-based diet, and had received recommended vaccinations. No antiepileptic treatment had been administered.

Upon presentation to Ars Veterinary Hospital, the dog was in good body condition, alert, and responsive to external stimuli. Physical and neurologic examinations revealed only a mild dysmetric gait. Either a multifocal or diffuse intracranial lesion was suspected that possibly involved the cerebrum and cerebellum. Differential diagnoses at that time included infectious encephalitis (e.g., canine distemper virus, Toxoplasma spp., bacterial infection), meningoencephalitides of unknown etiologies (e.g., granulomatous meningoencephalomyelitis, necrotizing meningoencephalitis), metabolic causes (e.g., portosystemic shunt, lysosomal storage diseases, organic acidurias), developmental anomalies (e.g., hydrocephalus), and degenerative encephalopathies (e.g., subacute necrotizing polioencephalopathy, lysosomal storage disease).

Initial diagnostic tests included hematology, serum biochemistry, electrolytes, bile acid stimulation test, and abdominal ultrasound. The serum biochemistry profile, hematology, and fasting and postprandial bile acid concentration results were all within reference ranges. The abdominal ultrasound examination did not reveal any evidence of hepatopathy or vascular anomaly.

MRI of the brain was performed^a after the dog was anesthetized with IV diazepam^b (0.5 mg/kg) and IV propofol^c (3 mg/kg), and maintained on isoflurane^d with 100% oxygen. T1-weighted, T2-weighted, and fluid-attenuated inversion recovery sequences (FLAIR) were obtained in sagittal, transverse, and dorsal planes. In addition, T1-weighted images were obtained following IV administration of gadolinium-DTPA^e. A sample of cerebrospinal fluid (CSF) was collected from the cerebellomedullary cistern following the MRI examination.

To measure urinary organic acids, 10 mL of urine were collected by cystocentesis, which was subsequently analyzed by gas chromatography-mass spectroscopy^f. Organic acids profiles were also quantified in serum and CSF in this case.

Genomic DNA was isolated from blood samples collected in ethylenediaminetetraacetic acid according to the instructions of the manufacturer^g. The complete open reading frame and the adjacent splices sites of the L2HGDH gene were amplified with polymerase chain reaction using the primers described by Penderis et al. (2007) and were analyzed by direct DNA sequence analysis.³ The primers were extended with M13 sequences, allowing sequencing with both M13 forward and reverse primers. The products were analyzed by capillary electrophoresis using the AI 3130xl genetic analyzer^h and assessed using Mutation Surveyorⁱ.

MRI revealed bilaterally symmetrical, diffuse regions of gray matter hyperintensity on T2-weighted and FLAIR sequences, which were most prominent in the parietal cerebral cortex, thalamus, mesencephalon, and cerebellum (Figure 1). A mild ventriculomegaly was also found in this case. Postcontrast T1-weighted sequences showed no contrast enhancement.

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Based on the MR imaging findings, differential diagnoses for bilaterally symmetrical polioencephalopathy included metabolic disease (hepatic encephalopathy, organic aciduria), nutritional deficiency (thiamine deficiency), neurodegenerative disorder (subacute necrotizing polioencephalopathy), or toxicosis (salt poisoning). Neoplasia and inflammation were considered to be far less likely. The distribution and appearance of the MRI findings were most suggestive of an inborn error of metabolism.

The urinary organic acid profile was quantified, which identified a >1,000-fold increase in 2-hydroxyglutaric acid (3,993 mmol/mol creatinine; reference range, 0.6–5.7 mmol/mol creatinine).² In addition, 2-hydroxyglutaric acid levels were also elevated in both serum (21.5 μmol/L; reference range, 0.4–4.2 μmol/L) and CSF (90 μmol/L; reference range, 0.1–1.4 μmol/L).² Enantiometric analysis confirmed the L-configuration, establishing the diagnosis of L2HGA.

Finally, genomic DNA sequence analysis of the complete open reading frame also confirmed the diagnosis of L2HGA. A mutation was detected disrupting the translation of the initiation codon of L2HGDH (c.1A>G; p.Met1?) as shown in Figure 2. No other variants were detected in the genomic DNA analysis. Unfortunately, pedigree information was not available for this study.

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Routine analysis of CSF demonstrated a normal nucleated cell count, morphology, differential count, and negative Pandy reaction.

Antiepileptic treatment was initiated with phenobarbitone^j (3 mg/kg per os q 12 hr) and rectal diazepam^k (0.5 mg/kg). After 3 wk of therapy, the serum phenobarbitone level was 21 μg/mL (therapeutic range, 15–35 μg/mL). Serum levels were measured q 3 mo and because clinical response was good, no modification to the original oral dose was recommended.

At the time this report was written (3 yr after initial presentation), the dog was still alive, but still had severe ataxia, a hypermetric gait, and head tremors. No seizure activity was noted

Discussion

Organic acidurias are characterized by the presence of abnormal organic acids as a result of an error in a metabolic pathway. L2HGA affects both humans and dogs and has an autosomal recessive mode of inheritance. The condition is characterized by increased levels of L-2-hydroxyglutaric acid in urine, CSF, and plasma.^1–3

L2HGA has three different clinical phenotypes in humans: neonatal without congenital anomalies, neonatal with congenital anomalies, and late onset.^4–6In the Yorkshire terriers described in this report, as in most human patients and the Staffordshire bull terrier, development of ataxia and seizures began within the first few years of life.^2,4,5

L2HGA was first reported in dogs by Abramson et al. in 2003. They reported L2HGA affecting six Staffordshire bull terriers that were presented for progressive neurologic signs.² Typically, those dogs presented between 6 mo and 1 yr of age, but affected dogs can initial present as late as 7 yr of age. Clinical signs included ataxia, muscular stiffness during exercise or excitement, altered behavior, and/or epileptic seizures.^1–3 In humans, L2HGA is characterized by a variety of neurologic signs, including psychomotor retardation, seizures and cerebellar ataxia.^4–7

In L2HGA, L-2-hydroxyglutaric acid accumulates secondary to a deficiency in flavin adenine dinucleotide (FAD)-linked L-2-hydroxyglutarate dehydrogenase, a mitochondrial enzyme, which usually converts L-2-hydroxyglutaric acid to α-ketoglutaric acid (an intermediate metabolite in the Krebs’s cycle). The neurologic signs in L2HGA are most likely a consequence of a toxic effect on the mammalian brain due to the accumulating metabolite, L-2-hydroxyglutaric acid. That metabolite has been shown to induce oxidative stress in the cerebral and cerebellar cortices and to inhibit mitochondrial creatine kinase, reducing brain energy metabolism in the rat.^8,9 In a study involving rat brains, the cerebellum appeared to be more vulnerable to the effect of L-2-hydroxyglutaric acid.⁷ This is an interesting observation because cerebellar involvement has been observed in dogs affected by L2HGA.^1–3

The combination of the MRI abnormalities (i.e., bilaterally symmetrical T2-weighted hyperintensity of the gray matter in the thalamus, basal nuclei, dorsal portion of the brainstem, and cerebellar nuclei) appears to be a common feature for L2HGA in dogs. Nonetheless, some of the individual characteristics can be found with other metabolic, toxic, and neurodegenerative encephalopathies.^1–3

In human patients, typical MRI findings of L2HGA consist of prominent T2-weighted hyperintensity of the subcortical white matter tracts and T2-weighted hyperintensity in the gray matter regions of the basal nuclei and dentate nucleus. The areas of T2-weighted hyperintensity often correlate with regions of hypointensity on T1-weighted images, which is consistent with increased water content of the affected tissues. The cerebellum is prominently involved in most cases.^4,5,10

In the two cases described herein, the distribution and appearance of the MRI findings (bilaterally symmetrical polioencephalopathy) were most suggestive of an inborn error of metabolism, particularly in view of the absence of inflammatory CSF changes and any other systemic metabolic abnormality. The mild dilation of the ventricles in case 1, which may also be seen as an incidental finding in some normal Yorkshire terriers, is of unknown significance.^11,12

Seizure activity has been reported with hydrocephalus in both humans and dogs, but in Case 1, it is unlikely that the etiology of the seizure activity was related to the hydrocephalus and it was considered normotensive.

Laboratory tests disclosed an increased L-2-hydroxyglutaric acid in different biologic fluids, making urine an adequate sample for initial diagnostics, until commercial DNA analysis is available for Yorkshire terriers.^13,14 Alternatively, (targeted) mutation analysis could be used to screen Staffordshire bull terriers.

In humans, up to 86 different gene mutations encoding for the L2HGDH gene have been identified on chromosome 14q22, which causes suppression of enzyme activity.^7,13 Some of those mutations result in amino acid substitutions in the L-2-hydroxyglutarate dehydrogenase. Other mutations either delete one or more amino acids from this enzyme. Regardless, those changes to the enzyme probably impair the normal function of L-2-hydroxyglutarate dehydrogenase by affecting how the enzyme folds into a three-dimensional shape. With a shortage of functional enzyme, L-2-hydroxyglutaric acid does not break down and instead accumulates in cells. Researchers believe that this accumulation is toxic and damages brain cells, leading to the signs and symptoms of L2HGA.^7,10

One recently published study reported that the detection rate of pathogenic variants in the L2HGDH gene for human patients with an elevated urinary excretion of L-2-hydroxyglutaric acid was 100%. In humans, an elevated urinary excretion of L-2-hydroxyglutaric acid can therefore be considered diagnostic for L2HGA.¹³

L2HGA is an autosomal recessive condition in the Staffordshire bull terrier and two single nucleotide polymorphisms (SNPs) have been identified in the homolog canine gene on chromosome 8 encoding for L-2-hydroxyglutarate dehydrogenase, which is responsible for the suppression of enzyme activity in affected animals. Both SNPs are on exon 10, and the mutation causes the substitution of two amino acids (from leucine and histidine to proline and tyrosine, respectively). Homozygosity for the minor allele of these SNPs correlates with L2HGA in Staffordshire bull terriers. Currently, there are no published reports of any clinical signs associated with heterozygosity.³

An additional study of Staffordshire bull terriers and 1,043 epileptic dogs of other known breeds demonstrated that two SNPs identified on exon 10 of the L2HGHD gene were specific to Staffordshire bull terriers.¹⁵ That study included 14 West Highland white terriers (but not Yorkshire terriers), and none of the included dogs had the SNPs in the L2HGDH gene. This supports the contention that L2HGA can be caused by more than one mutation, as has previously been reported in humans.

A genetic mutation investigation of the two dogs in this study revealed the absence of the two above mentioned SNPs that occur in Staffordshire bull terriers, but revealed another homozygous mutation on exon 1. This mutation involved a substitution of the nucleotide adenine for guanine, which changed the amino acid sequence (from methionine to valine) of the translation initiation codon.

The c.1A>G mutation has been detected previously in human patients affected with L2HGAⁿ. This mutation reportedly affects the translation initiation site.¹³ The translation initiation codon is generally defined as the point or sequence, at which a ribosome begins to translate a sequence of RNA into amino acids. The initiation codon is typically AUG (or ATG in DNA), encoding methionine.¹⁶ The c.1A>G is an interesting mutation as its effect cannot be exactly predicted. That mutation could result in either the absence of protein or alternatively another translation initiation site may be used (upstream or downstream) that may result in an alternative protein. The same mutation was identified in one human patient, a 12 mo old girl. Similar clinical signs (epilepsy, cerebellar ataxia, and developmental delay) existed as described in the dogs in this report.¹³

The clinical presentation and MRI features of L2HGA in the Yorkshire terriers in this report were consistent with those previously reported in the Staffordshire bull terrier and West Highland white terrier.^1,2 The mutation is different than that seen in Staffordshire bull terriers.

Currently, no specific treatment is available for L2HGA in affected humans or dogs. Case reports in humans have described positive effects of administering FAD in combination with levocarnitine chloride in one patient and riboflavin, a precursor of FAD, in another patient.^6,17 Either the complete loss of function or a defective L-2-hydroxyglutarate dehydrogenase (which is a FAD-dependent enzyme) is responsible for the accumulation of L-2-hydroxyglutaric acid in body fluids. Therefore, it might be expected that FAD, riboflavin, and/or levocarnitine could enhance enzymatic residual activity.¹⁷

In the two dogs with seizure activity in this report, phenobarbitone (3 mg/kg/day) adequately controlled seizures. Other anticonvulsants, such as levetiracetam, have been used in addition to phenobarbitone and potassium bromide to treat recurrent and cluster seizures due to an organic aciduria in a dog.¹⁸ Because the numbers of reported cases of organic acidurias is very low, the prevalence of seizure activity in affected animals and the success of seizure therapy are unknown, but deserve further investigation.

Conclusion

This reported describes, for the first time, L2HGA in Yorkshire terriers. The mutation causing L2HGA in Yorkshire terriers is different than the one noted in Staffordshire bull terriers. Affected dogs have dramatically increased levels of L-2-hydroxyglutaric acid in urine and have characteristic MRI findings (i.e., bilaterally symmetrical, diffuse regions of gray matter hyperintensity on T2-weighted and FLAIR sequences). The biologic hallmark of L2HGA is the finding of increased levels of L-2-hydroxyglutaric acid in a variety of tissues and fluids, including urine. Until DNA testing is widely available for Yorkshire terriers, L2HGA can be diagnosed by assessment of urinary organic acids, which should be performed in all patients with clinical and/or neuroimaging findings suggestive of this disease.

As a future prospect, the understanding of the function of L-2-hydroxyglutaric acid would be particularly valuable to understand the pathophysiology of the disease. A better understanding of the disease mechanism will hopefully allow for the development of therapeutic strategies for L2HGA.