Identification of novel L2HGDH mutation in a large consanguineous Pakistani family- a case report

Background L-2-hydroxyglutaric aciduria (L2HGA) is a progressive neurometabolic disease of brain caused by mutations of in L-2-hydroxyglutarate dehydrogenase (L2HGDH) gene. Cardinal clinical features include cerebellar ataxia, epilepsy, neurodevelopmental delay, intellectual disability, and other clinical neurological deficits. Case presentation We describe an index case of the family presented with generalised tonic-clonic seizure, developmental delay, intellectual disability, and ataxia. Initially, the differential diagnosis was difficult to be established and a SNP genome wide scan identified the candidate region on chromosome 14q22.1. DNA sequencing showed a novel homozygous mutation in the candidate gene L2HGDH (NM_024884.2: c.178G > A; p.Gly60Arg). The mutation p.Gly60Arg lies in the highly conserved FAD/NAD(P)-binding domain of this mitochondrial enzyme, predicted to disturb enzymatic function. Conclusions The combination of homozygosity mapping and DNA sequencing identified a novel mutation in Pakistani family with variable clinical features. This is second report of a mutation in L2HGDH gene from Pakistan and the largest family with L2HGA reported to date.

Background L-2-hydroxyglutaric aciduria (L2HGA) is a rare autosomal recessive neurodegenerative disorder of metabolism [OMIM #236792] which is due to the accumulation of L-2-hydroxyglutaric acid (LGA) in urine, plasma and cerebrospinal fluid (CSF) [1,2]. The phenotypic features of this organic aciduria are diverse, including developmental delay, cerebellar ataxia, epilepsy, severe intellectual disability, and macrocephaly [3][4][5]. The onset of disease has been reported to occur at an early age with severe epileptic fits or neurodegenerative symptoms, although it may also appear in adulthood with less severe presentations. There are reports of increased incidence of the development of brain tumours due to progression in L2HGA [6][7][8][9].
The present case describes the clinical presentation and mutation analysis of L2HGDH in a large Pakistani consanguineous family comprising multiple individuals affected by a metabolic neurological disorder. Homozygosity and sequencing studies revealed a rare missense mutation (NM_024884.2:c.178G > A; p.Gly60Arg), in exon 2 of L2HGDH as the likely cause of disease in this family.

Case presentation
A 16 year old girl (IV-4) presented to hospital with history of seizures since the age of 8 months, intellectual delay, and ataxia. At the age of 13 years she was described by parents as 'mentally dull' and generalized tonic-clonic seizures recurred with increased frequency, mostly at night. Her elder sister (IV-6) and brother (IV-1) also showed symptoms of epilepsy and intellectual delay, as did three first cousins (IV-8, IV-9, and IV-10) although no in depth clinical evaluation was performed.

Clinical evaluation of index case
On physical and clinical examination IV-4 was alert, with an ataxic gait. Manual muscular testing did not note any weakness of limbs, but mild finger and nose ataxia was apparent along with a retarded capability of speech. Her deep tender reflexes were +++ and symmetrically preserved, while the plantar responses were bilaterally flexor. Her brain MRI showed abnormal diffuse T2 hyperintense signals in the subcortical white matter and bilateral symmetrical T2 hyperintense signals in bilateral basal ganglia (Fig. 1). Mild cortical cerebellar atrophy was also seen. Electroencephalogram (EEG) examination showed moderate diffuse encephalopathy/moderate diffuse brain dysfunction and observed epileptiform activity arising from the right hemisphere ( Fig. 1). Urine testing for L2-hydroxyglutarate was not possible due to unavailability of this test in regional diagnostic laboratories, and remote setting of the family involved. Genetic investigation was recommended and she was advised for tablet Neurobion 1 BD, tablet Folic acid 5 mg OD, capsule Coenzyme Q-10, 50 mg BD, tablet Loprin 75 mg for symptomatic management.

Molecular genetic analysis
Genomic DNA was extracted from peripheral blood samples. Due to the availability of multiple affected family members and the consanguinity of the parents of the index case (Fig. 1a), autozygosity mapping through genome-wide SNP genotyping was conducted as previously described using Illumina Human CytoSNP-12 v2.1 microarrays [20]
On structural analysis, the Gly60 residue of L2HGDH resides in the helix region. This short non-polar glycine residue is replaced in the mutant protein by the larger, more positively charged and hydrophilic arginine residue. The Gly60Arg mutation is predicted to produce a minor local conformational change due to the difference in the observed contacts and surface area. The native glycine residue is only involved in intermolecular interactions with threonine at position 90 while the replaced basic arginine introduces an electrically charged, basic guanidium group which, unlike glycine, has more hydrogen bonding capabilities leading to the formation of an a inter molecular hydrogen bond with Thr90 as well as with Arg196 and Thr195. This leads to a slight local perturbation of the helix conformation for mutated protein (Fig. 3).

Discussion and conclusions
In this study, we describe a large pedigree from Pakistan showing multiple neurological symptoms. Homozygosity mapping and Sanger sequencing revealed a novel missense mutation in L2HGDH gene. This is the second report of L2HGDH mutation in a Pakistani family; previously a nonsense mutation (p.Arg335Ter) was reported in a family showing a neuro-degenerative disorder of metabolism with two affected individuals [8]. Clinical and radiologic examinations of affected individuals identified presence of a slowly progressive neurodegenerative disease with cerebellar ataxia, seizures, delay in growth and abnormal subcortical white matter. MRI showed the persistent changes in the subcortical white matter characteristic in L2HGA leukoencephalopathy while the brain stem involvement in other leukoencephalopathy [11,15]. Although additional phenotypic characteristics are described in the literature, including macrocephaly, pyramidal and extra-pyramidal features, these were not present in our patients, [15,21].
L2HGDH encodes L-2 hydroxyglutare dehydrogenase which is the key contributor for this neurodegenerative disease. A large number of families and cases are reported with more than 100 pathogenic mutations in this gene. These mutations are mostly repeated in different ethnic populations. Interestingly, the disease is mostly reported in families from Mediterranean origin with numerous families reported from Turkey, Tunisia, Italy and Lebanon [6,8,15,17]. Currently, there are 162 families with 283 cases which have been investigated for mutations in L2HGDH gene comprising a total of 112 mutations, 36 of which are found repeatedly found in different ethnic groups (http://grenada.lumc.nl/LOVD2/ vumc/status.php).
The possible impact of our mutation on protein function was investigated using in silico bioinformatics tools. The mutation was predicted to affect the hydrogen bonding, and thus alter the stereochemistry of the protein (Fig. 3) [22].
To conclude, this case report provides the molecular diagnosis of a large consanguineous Pakistani family with six individuals. We identified a novel L2HGDH mutation predicted to cause in a loss of stability of L2HGDH protein.