Gamma-aminobutyric acid (GABA) being the major inhibitory neurotransmitter in the CNS has key role in modulating neuronal activity. GABAergic neurons are widespread in CNS and play key role in controlling epileptic foci. A reduced pre-synaptic GABAergic inhibition of D2 dopaminergic neurons, via GABA-A receptors can cause epileptic seizures. Different epileptic syndromes are associated with different subunit genes mutations including GABRA1, GABRB3, GABRD & GABRG2 [1]. The gene encoding for B3 subunit of GABA-A is located on chromosome 15q11.2-q12 whose reduced expression is involved in causation of a spectrum of disorders like absence seizures, Angelman Syndrome, autism spectrum disorders etc. [2-4]. Recently the mutations in GABRB3 were also identified in cases of infantile spasms and Lennox Gestaut Syndrome. Till date only a few cases of GABRB3 mutation causing early infantile developmental and epileptic encephalopathy (EIDEE) have been described in literature.

Clinical spectrum of Biotinidase deficiency includes hypotonia, seizures, eczematous skin rash, respiratory problems, developmental delay, hearing and vision problems. Metabolically, most untreated individuals will have ketolactic acidosis, organic aciduria and mild hyperammonemia.

Here we report a case of early infantile epileptic encephalopathy with GABRB3 mutation with biotinidase deficiency and G6PD deficiency identified on a diagnostic genetic panel.

A male child 5th born to non-consanguineous parents, was delivered by normal vaginal delivery at 40 weeks of gestation. The antenatal and perinatal period was unremarkable. Family history was non-contributory, all siblings were normal. The patient presented at 2 months of life with feeding difficulties and convulsions which were of generalized tonic clonic type. Seizures gradually increased in frequency. He was progressively added with multiple anti-convulsants (Initially Phenobarbitone, Phenytoin, Valproate, Levetiracetam with Biotin and clobazam and finally midazolam infusion and ketogenic diet. But despite all medications, he continued to have intractable epilepsy. During his admission at our hospital (at 3 months of age), he had 15-20 epileptic seizures per day of varied semiology, including GTCS, focal motor events, myoclonic jerks and brief tonic seizures.

Over the time, child lost his achieved milestone that is social smile and neck holding. Head circumference was normal for age. A few subtle dysmorphic features were evident in form of tented mouth appearance, long eyelashes with light brown, sparse hair, dry skin. Child was hypertonic, deep tendon reflexes were brisk and plantar response extensor bilaterally with ankle clonus. Other systems were within normal limits (Figure 1).

Figure 1(a-b): (a) Clinical Photograph of Patient Before Starting Treatment Showing Sparse Hair and Dry Skin and (b) Clinical Photograph Showing Improvement in Hair Growth and Skin Texture After Biotin Treatment

Figure 2: EEG of the patient

EEG was done which showed focal slowing and epileptic activity in right occipital region (Figure 2). CT scan & MRI brain was done at the age of 3 months which were normal. Extensive neurometabolic investigations were done including serum lactate, ammonia levels, CSF glycine levels, GCMS-TMS which were essentially normal. Serum Biotinidase levels were sent in view of phenotypic resemblance and intractable epilepsy which came deficient with biotinidase value of 0.9 mmol (Range-4.1-14.5). Clinical exome showed mutation in GABRB3 gene coding for beta-3 subunit of GABA-A. Patient was gradually weaned off intravenous anti-convulsants and disharged when seizure free on four oral anti-convulsants with biotin supplemetation. In follow-up visits, patient showed gross developmental delay with intermittent episodes of seizures with above mentioned semiology but in a far lesser frequency. There was significant improvement in skin and hair changes patient have had before.

Genetic Investigation

Clinical exome was performed covering 8000 genes. Sequencing of the protein coding regions in genes associated with inherited diseases was performed using illumina next generation sequencing at a mean coverage of 80-100X in the target region. Segragation of the variant could not be carried out due to monetary restrictions of the family. A heterozygous missense variant (C. 860 C>T) in exon 8 of the GABRB3 gene that results in amino acid substitution from threonine to isoleucine at codon287 (p.Thr 287 Ile) was identified. There is moderate physicochemical difference between threonine and Isoleucine. This variant has been classified as likely pathogenic according to American college of medical genetics guideline with autosomal dominant inheritance.

The gamma-aminobutyric acid (GABA) type A receptor B3 gene (GABRB3) encodes the b3-subunit of GABA Type A receptor which is primary mediator of inhibitory synaptic transmission in the central nervous system. A mutation in GABRB3 receptor with Biotinidase deficiency can be the cause of severe intractable early infantile developmental and epileptic encephalopathy in our patient. Biotinidase deficiency is a pre-established cause & GABRB3 is an emerging cause of early onset epilepsy syndromes, their concomitant existence has not been reported to our knowledge. GABA is the major inhibitory neurotransmitter in the CNS. Its action is mediated via two types of receptors-Ionotropic GABA-A & GABA-C and metabotropic GABA-B receptors. Out of which GABA-A & C form chloride channels and take part in fast synaptic inhibition, while GABA-B receptors are G-Protein binding receptors that modulate calcium & potassium channels and cause both presynaptic & slow post-synaptic inhibition.

Very few cases of GABRB3 mutation epileptic encephalopathy have been described in literature. Our case of early infantile epileptic encephalopathy with GABRB3 mutation with co-existent biotinidase deficiency. The spectrum of clinical features of GABRB3 mutation and biotinidase deficiency are similar in initial phase, while GABRB3 mutation may show varied symptoms in later childhood due to its association with genes causing autism, rett syndrome, angelman syndrome etc. Although, the incidence of their concomitant existence and whether it causes any pathogenic synergism, will require further research. Though adding biotin supplementation showed significant improvement in the skin and hair changes, seizures never got satisfactorily controlled even on the highest dosages, signifying the presence of some other or additional underlying mechanism, which in our case is attributable to GABRB3 mutation.

To the date, 27 other cases of GABRB3 EIDEE have been reported [5,6] and no case with co-existing biotinidase deficiency is reported to our knowledge. The features that are common in these cases include: (1) Onset of seizures in early infancy, (2) Varied typed of seizures and (3) Variable EEG abnormalities.

Our patient had a mutation affecting neurotransmitter gated ion channel in transmembrane region of GABRB3 HUMAN protein which is rare as most of the mutations target the extracellular protein domains near the amino (N) terminus [7]. The mechanism of pathogenicity can be possibly due to change of amino acid position within the receptor pore which could negatively impact function of the receptor. The phenotypic spectrum of GABRB3 mutation ranges from simple febrile seizures, genetic epilepsy with febrile seizures plus, myoclonic- atonic seizures to west syndrome and other severe early onset epileptic encephalopathy. Neurological disorders like Rett syndrome (deficiency of MeCP2), Angelman syndrome and autism exhibit reduced expression of GABRB3 and UBE3A [8,9]. Beta-3 subunit of GABA receptor is earliest appearing in the embryonic brain. It is expressed at 150% of adult levels in perinatal brain, most prominently in thalamus. Point mutations in peptide coding region of exon 1A of GABRB3 correlates with childhood absence epilepsy. Same signal variant also provides link between GABRB3 mutation and autism spectrum disorder which explains seizures in ASD [10].

Biotinidase is the enzyme that recycles biotin. In have two types of activities- biotinyl hydrolase and biotinyl transferase. Biotinides is a mass of 76-77 KDa. It has atleast 9-isoforms. Biotinidase processes protein bound biotin in diet. It transfers biotin from biocytin to nucleophiles. The gene for human biotinidase (BTD) lies on chromosome 3q25. Biotinidase deficiency presents with varies symptoms like hypotonia, ataxia, seizures, dermatitis, alopecia and recurrent respiratory infections.

Research is going on mechanisms of GABRB3 affecting neuronal networks causing epilepsy and neurodevelopmental delay. In conclusion, GABRB3 gene mutation along with Biotinidase deficiency can cause refractory epilepsy and their simultaneous occurrence in a patient is rare and further research is needed to identify pathogenesis, novel therapies and wider implications of the mutations in other neurological disorders.

This is a case report of early infantile developmental and epileptic encephalopathy with GABRB3 mutation which is associated with multiple neurodevelopmental disorders. This report underlines the importance of genetic testing in intractable epilepsy.

Acknowledgment

The authors thank Dr. Mohan Joshi, Dean-T.N. Medical College & BYL Nair Hospital for granting permission to publish this manuscript.

1. Baumann, S.W. et al. “Forced Subunit Assembly in α1β2γ2 GABAA Receptors: Insight into the Absolute Arrangement.” Journal of Biological Chemistry, vol. 277, no. 48, 2002, pp. 46020-46025.

2. Bowser, D.N. et al. “Altered Kinetics and Benzodiazepine Sensitivity of a GABAA Receptor Subunit Mutation [γ2(R43Q)] Found in Human Epilepsy.” Proceedings of the National Academy of Sciences, vol. 99, no. 23, 2002, pp. 15170-15175.

3. Macdonald, R.L. et al. “Mutations in GABAA Receptor Subunits Associated with Genetic Epilepsies.” The Journal of Physiology, vol. 588, no. 11, 2010, pp. 1861-1869.

4. DeLorey, T.M. et al. “Mice Lacking the β3 Subunit of the GABAA Receptor Have the Epilepsy Phenotype and Many of the Behavioral Characteristics of Angelman Syndrome.” Journal of Neuroscience, vol. 18, no. 20, 1998, pp. 8505-8514.

5. Papandreou, A. et al. “GABRB3 Mutations: A New and Emerging Cause of Early Infantile Epileptic Encephalopathy.” Developmental Medicine & Child Neurology, vol. 58, no. 4, 2016, pp. 416-420.

6. Møller, R.S. et al. “Mutations in GABRB3: From Febrile Seizures to Epileptic Encephalopathies.” Neurology, vol. 88, no. 5, 2017, pp. 483-492.

7. Ernst, M. et al. “Comparative Models of GABAA Receptor Extracellular and Transmembrane Domains: Important Insights in Pharmacology and Function.” Molecular Pharmacology, vol. 68, no. 5, 2005, pp. 1291-1300.

8. Tanaka, M. et al. “GABRB3, Epilepsy and Neurodevelopment.” Epilepsia, vol. 51, 2010, p. 77.

9. Hirose, S. “Mutant GABAA Receptor Subunits in Genetic (Idiopathic) Epilepsy.” Progress in Brain Research, vol. 213, 2014, pp. 55-85.

10. Delahanty, R.J. et al. “Maternal Transmission of a Rare GABRB3 Signal Peptide Variant Is Associated with Autism.” Molecular Psychiatry, vol. 16, no. 1, 2011, pp. 86-96.