BCKDK gene mutations as a rare condition responsible for comorbid neurodevelopmental delay, autism, and epilepsy: a case series of four patients

Abstract

Introduction:

Mutations in the branched-chain keto-acid dehydrogenase kinase gene (BCKDK), leading to low plasma branched-chain amino acids (BCAAs) levels, have been reported as a contributor to comorbid intellectual disability, autism, epilepsy, and neurodevelopmental delay (NDD). Due to the rarity of knowledge about these mutations, the current case series aims to introduce four confirmed cases.

Methods:

This case series study analyzed children from a neurometabolic clinic. Social and adaptive functions were assessed using the vineland social maturity scale (VSMS). Whole exome sequencing (WES) identified genetic variants filtered using population databases. Candidate variants were confirmed through Sanger sequencing and interpreted based on ACMG guidelines.

Results:

Four children of unrelated consanguineous families suffering from global NDD and autism were referred to our center. Neuroimaging assessments revealed negligible findings; thus, metabolic tests were sent, in which BCAAs were lower than normal limits. Therefore, genetic testing was done, and genetic variants compatible with BCKDK deficiency were detected. By initiating a BCAAs-rich regimen, the patients had significant improvements in psychomotor and speech development.

Conclusion:

The diagnosis of BCKDK deficiency should be suspected in patients with NDD and autism, and BCAA supplementation should be initiated as soon as diagnosis confirmation to prevent irreversible brain damage. The results emphasize that early diagnosis and dietary intervention by regulating plasma BCAA levels lead to the prevention of irreversible neurodevelopmental implications.

Introduction

The incidence of inherited metabolic disorders (IEMs) is individually rare (< 1:100 000), but considering the diversity of the enzymatic pathways, the prevalence of these disorders is estimated to be 1:1000 in Iran^[1]. A detailed metabolic reassessment has identified the causative IEM in up to 14% of patients previously classified as having global developmental delay of unknown origin^[2].

HIGHLIGHTS

• Mutations in the BCKDK gene lead to low plasma branched-chain amino acids (BCAAs), contributing to neurodevelopmental delay (NDD), epilepsy, autism, and intellectual disability.

• This case series reports four children from consanguineous families diagnosed with BCKDK deficiency, all exhibiting global NDD, language impairment, and, in some cases, seizures.

• Genetic testing confirmed BCKDK mutations in all patients after metabolic assessments showed decreased BCAA levels, despite normal neuroimaging findings.

• Treatment with a BCAA-rich diet led to significant improvements in psychomotor and speech development, highlighting the importance of early dietary intervention.

• The study underscores the need for metabolic screening in NDD patients and timely BCAA supplementation to prevent irreversible neurological damage.

Studies show that 18% of all currently known IEMs are treatable^[3], and branched-chain keto-acid dehydrogenase kinase (BCKDK) deficiency is considered one of these treatable IEMs^[4].

Mutations in the BCKDK gene have been reported as a contributing cause of comorbid intellectual disability, epilepsy, autism, and microcephaly. Low plasma branched-chain amino acids (BCAAs) levels are the other characteristic of these cases^[5]. BCAAs, including leucine, isoleucine, and valine, are essential amino acids and are only stored in the form of protein. BCAAs constitute approximately 20% of the incorporated amino acids into proteins. Accordingly, the exceeded BCAAs must be removed via a catabolic pathway of deamination followed by the irreversible oxidative decarboxylation of the deaminated products to their corresponding acyl-CoA esters. This process is done by a mitochondrial branched-chain amino acid aminotransferase and the branched-chain α-keto acid dehydrogenase complex (BCKD)^[6]. BCKD is a protein that is composed of four catalytic subunits, including E1α, E1β, E2-DBT, and E3-DLD^[7], which are encoded by four different genes. Accordingly, due to the similarity of this protein with the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes, this complex can bind to this strategic point that plays a critical role in the regulation of BCAA metabolism, which means a careful regulation of its activity is crucial in correct BCAA metabolism^[8]. Phosphorylation is the pathway to control the BCKD complex activity, which is performed by BCKDK and is influenced by dietary and hormonal stimulators^[9]. When plasma BCAA levels are decreased, BCKD turns to the inactive form through phosphorylation^[10]. Impaired plasma BCAA levels cause widespread disruption in organ function, including the heart^[11], liver^[12], skeletal muscles^[13], and especially the brain, as it easily crosses the blood-brain barrier (BBB) and is crucial for the maintenance of brain function^[14].

The mentioned pathway, as well as identifying some patients with the presentations of neurodevelopmental delay (NDD) who have been found to have low plasma BCAA levels, has ignited the idea about the role of BCKDK deficiency in this phenotype^[15]. The current study presents four Iranian children with NDD who were clinically and genetically evaluated and represented BCKDK gene mutations responsible for their condition.

Methods

This study was conducted as a case-series study and followed the Preferred Reporting of Case Series in Surgery (PROCESS) guideline^[16]. Data were collected from children admitted to our neurometabolic outpatient clinic between December 2018 and July 2020. For the evaluation of social and adaptive functions, Vineland Social Maturity Scale (VSMS) was used^[17].

Genetic investigation

Genomic DNA was extracted from the patient’s blood sample using a commercial purification kit (GeneAll®, Korea). The concentration of the purified DNA was measured using a Thermo Scientific™ Nanodrop 2000.

Whole Exome Sequencing (WES) was performed by CeGaT GmbH (Germany). All exons were captured using the Twist Human Core Exome Kit (Twist Bioscience, USA), and the captured DNA was then sequenced using the Illumina NovaSeq 6000 platform (Twist Bioscience, USA).

After sequencing, the raw data was processed to check its quality and then aligned to the human reference genome 19 (GRCh37/hg19) using Burrows-Wheeler Aligner (BWA). In order to perform indel realignment and base recalibration, Assembly Based ReAligner (ABRA) [version 2.18] was applied. Variant calling was done using the Genome Analysis Tool Kit (GATK) HaplotypeCaller program. These variants were then annotated with the ANNOVAR program.

To prioritize disease-causing variants, a stepwise filtering approach was used. Variants that were common in the general population were excluded based on MAF > 0.05 in dbSNP147 (https://www.ncbi.nlm.nih.gov/SNP/), 1000 Genomes Project (https://www.internationalgenome.org/), Exome Aggregation Consortium (ExAC) (http://www.Exac.broadinstitute.org/), Genome Aggregation Database (gnomAD) (https://gnomad.broadinstitute.org/), the Iranome catalog of genomic variants in the Iranian population with 800 healthy individuals, and the in-house database of Iranian exomes (Watson Genetic Laboratory, Iran). Only variants located in protein-coding regions or splicing sites were considered.

Candidate genes associated with the patients’ condition were identified using medical literature databases like OMIM (Online Mendelian Inheritance in Man) and PubMed. The pathogenicity of each variant was evaluated using additional databases such as ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and HGMD (https://www.hgmd.cf.ac.uk/ac/index.php).

Finally, the interpretation of the candidate variants was performed according to guidelines established by the American College of Medical Genetics and Genomics (ACMG).

Segregation analysis and variant confirmation

To confirm the identified variants and analyze their inheritance pattern within the family, specific DNA primers were designed to amplify the region of the gene containing the variants. PCR-Sanger sequencing (ABI 3500 Genetic Analyzer) was performed on the patients’ sample and their parents. Then the sequencing results were aligned to the reference sequence using Chromas software (version 2.6.2).

Results

Patient 1

A 4-year-old girl was referred to our neurometabolic clinic with NDD and agitation. She was the first child of a consanguineous marriage who had a normal body birth weight (BBW = 2400 g). Motor development was delayed. By 9 months of age, she could hold her neck. She could walk some steps at around 2 years of age, and now she can walk independently, but her speed, balance, and coordination were impaired. Language is also impaired, and she can speak only a few words. She could speak some words at 4 years of age. The only reported family history of intellectual disability was a third-degree family member. The current body weight, head circumference, and biparietal diameter were 10 kg, 45 cm, and 33 cm, respectively. The auditory brainstem response (ABR) test, brain computed tomography, and magnetic resonance imaging (MRI) revealed normal findings. In the next step, metabolic tests were sent that presented low plasma BCAA levels, and lactate and free fatty acids were within normal limits (Fig. 1). Accordingly, WES was performed. Following analysis, a homozygous variant at the splice acceptor site of intron 3 of the BCKDK gene (NM_005881.4: c.265-2A>G) was detected (Table 1). Patient’s parents were first cousins and were heterozygous for the variant in this gene. Accordingly, a regimen containing BCAA supplementation was initiated, and significant improvement in the child’s psychomotor and language development was detected. She can speak some sentences and climb the stairs without help. VSMS score before and 2 years after treatment with a high-protein diet and BCAA supplements is shown in Fig. 2.

Figure 1.

Plasma BCAA levels before and after BCAA-rich regimen. The post-treatment test is not done by patient number 1 and 3.

Table 1.

Features of the variants found in the BCKDK gene (NM_005881.4)

Patient number	Variant	Exon/Intron	Allele frequency	Zygosity	Type and classification
1	c.265-2A>G	Intron 3/11	gnomAD: N/A Iranome: N/A	Homozygous	Canonical splice site, likely pathogenic
2	c.466C>T p.R156*	exon 6/12	gnomAD: 2 Hetro & 0 Homo Iranome: N/A	Homozygous	Nonsense, pathogenic
3	c.283C>T p.Q95*	exon 4/12	gnomAD: N/AIranome: N/A	Homozygous	Nonsense, likely pathogenic
4	c.823G>C p.Glu275Gln	exon 9/12	gnomAD: N/A Iranome: N/A	Homozygous	Missense, variant of uncertain significance

Figure 2.

VSMS score before and 2 years after BCAA-rich regimen. The post-treatment evaluation is not done by patient number three.

Patient 2

The second patient is a 40-month-old boy of consanguineous marriage with the chief complaint of NDD and agitation. His developmental delay was not detected until 5 months of life, and at that time an MRI was performed, which revealed normal findings. He could sit without help by 1 year of age and started to speak 3-4 words when he was three years old. Besides, his mother stated some courses of staring episodes, which have been well-controlled with primidone and clobazam. Considering the normal neuroimaging results, metabolic tests, including free fatty acids, serum lactate, and high-performance liquid chromatography, were administered, and it was shown that BCAAs were lower than the normal limits (Fig. 1). Further investigation by WES revealed a homozygous variant in exon 6 (c.466C>T, p.R156*) compatible with BCKDK deficiency (Table 1). Therefore, an amino acid-rich plus BCAA supplementation regimen was initiated and led to a significant improvement in agitation; the patient started to walk independently, and the seizures have been controlled completely. VSMS score before and two years after treatment with a high-protein diet and BCAA supplements is shown in Fig. 2.

Patient 3

An 8-year-old girl who was the first child of a consanguineous marriage was referred with NDD to our neurometabolic clinic. Psychomotor delay was noted within the first months of life. She walked when she was 30 months old. Currently, she can walk independently, but her coordination is impaired. Language delay is also detected, and she can speak only some limited words now. Besides, recurrent seizures developed. An MRI was performed in which mild thinning of the corpus callosum was detected while the other parts of the brain were normal. Further metabolic studies revealed BCAAs deficiency (Fig. 1). WES represented two mutations, one on the ZNF423 gene, a well-known one for Joubert syndrome, and another one that was a novel gene mutation in the BCKDK gene (c.283C>T, p.Q95*), compatible with BCKDK deficiency (Table 1). Although a BCAA-rich regimen was initiated, the parent was not adherent to the medications, and the severity of clinical seizures and neurodevelopmental status of the patient did not change.

Patient 4

A 13-year-old autistic girl of consanguineous marriage who was delivered through cesarean sections was referred to our neurometabolic clinic due to aggression. Her parents reported psychomotor delay by the first months of her life. Walking started at 22 months. Speaking is limited to some words. One episode of generalized tonic-clonic seizure was detected as well. Neuroimaging revealed nothing abnormal; however, the child had microcephaly. Metabolic tests were performed in which BCAAs were lower than normal limits (Fig. 1). The WES results revealed the presence of a homozygous missense variant defined as c.823G>C (p.Glu275Gln) in exon 9 of the BCKDK gene (Table 1). The patient’s parents were heterozygous for this variant. This sequence change replaces glutamic acid with a glutamine residue at codon 275 of the BCKDK protein. By the initiation of the BCAA-full regimen and levetiracetam for seizure control, improvement in development was detected. Her mother represented improvement in speech, and she could speak some sentences. There was no episode of seizure recurrence. VSMS score before and 2 years after treatment with a high-protein diet and BCAA supplements is shown in Fig. 2.

Discussion

The current study represents four children with NDD, agitation, and seizures. Neuroimaging assessments were normal, and the metabolic process was suspected, so diverse metabolic parameters were studied. Eventually, the genetic test was performed to assess BCKDK, which revealed causative variations in the BCKDK gene. Accordingly, by the initiation of BCAAs-rich supplementations, the language and psychomotor developmental patterns of the patients were improved. Due to the rarity of knowledge about the frequency and patterns of BCKDK gene mutations and their related presentations, this study tries to demonstrate a thorough vision.

The first description of the BCKDK gene mutation in humans as a cause of comorbid intellectual disability, autism, and epilepsy was in 2012 by Novarino et al, who reported three affected siblings from three consanguineous families^[5]. Similarly, García-Cazorla et al studied two patients with psychomotor delay and microcephaly. The remarkable decrease in the plasma BCAA levels, while the other amino acids were normal, drew attention to performing biochemical genetic tests, which showed mutations in BCKDK^[8]. Boemer et al reported three patients from an inbred family with BCKDK deficiency presenting with severe encephalopathy, autistic behavior, and poorly controlled seizures. During 18 months, they were treated with a protein-rich diet and BCAA supplements, which improved the neurological features of the patients^[18]. The normalization of plasma BCAA levels is the key to improving the global neurological function of these patients, which is in line with our study. Also, similar to this study, all patients with a mutation in the BCKDK gene, with one exception, in the study of García-Cazorla et al were from consanguineous marriages. Notably, one of the first studies assessing the effect of BCKDK−/− on the neurodevelopment of mice represented a combination of neurological presentations, including tremors, seizures, retarded growth, and motor deficit^[19].

These results can be explained by the fact that BCAAs are involved in vital cellular functions of the brain, such as protein synthesis, glutamate metabolism, and the generation of energy. BCAAs are essential amino acids, which approximately one-third of glutamate in the brain contains nitrogen from BCAAs^[20]. Net nitrogen transfer from BCAA to glutamate occurs when the carbon skeleton of the BCAA is irreversibly lost via the BCKD^[6,20]. As a glutamate is a major excitatory neurotransmitter that is essential for brain function maintenance. BCAAs are also necessary for the production of gamma-aminobutyric acid, a key inhibitory neurotransmitter that is produced by the decarboxylation of glutamate^[21]. Additionally, there is competition among plasma amino acids for transport through the BBB. A reduction in plasma BCAAs in BCKDK knockout mice has been shown to impact the transport of other large neutral amino acids, which are precursors for other important neurotransmitters, as well as human brain concentrations of BCAAs^[19,22]. Further, secondary altered mitochondrial ultrastructure, bioenergetics depletion, and mitochondrial dysfunction have been seen in fibroblasts derived from patients with BCKDK mutation^[23,24]. As a result of low plasma BCAA levels, the majority of patients with BCKDK gene mutations exhibit neurodevelopmental delays as well as problems in social, behavioral, and communication interactions. Also, inappropriate synaptic communications during the process of neural development explain the fact that up to 50% of patients with neurodevelopmental problems have, to some extent, the autistic features^[10].

However, these symptoms can be induced due to diverse etiologies, which lead to delayed or misdiagnosis of metabolic disorders. Therefore, in order to diagnose and treat patients with neurodevelopmental abnormalities, metabolic examination is essential. NDD is the key symptom of IEMs, which is often global, progressive, and associated with other neurological disorders^[25]. Metabolic inconsistency and increased mortality and morbidity are common between inborn errors in the BCAA pathways, while there are currently few treatment options available, mainly dietary management^[26]. Timely and accurate metabolic evaluation enhances quality of life and long-term prognosis in patients with neurodevelopmental disorders by providing personalized treatment plans^[27]. Although patients’ plasma BCAA levels may normalize with BCAA-enriched treatment, the therapy’s late initiation does not lead to complete brain BCAA restoration and only results in modest clinical improvements^[5].

The present results suggest blood amino acid concentration testing to detect possible BCKDK deficiency in newborns with NDD. However, several limitations must be acknowledged, such as the small sample size and lack of long-term follow-up. Also, non-adherence to the treatment, as in this study, led to poor follow-up in some patients, which can confound treatment response and should be investigated. Further studies with long follow-up in diverse populations should be conducted.

Conclusion

In summary, the current study represented four unrelated patients with neurodevelopmental delay in which BCKDK deficiency was diagnosed, and response to medication and BCAA supplementation was detected. Despite the rarity of BCKDK mutations, they should be notified as one of the probable causes responsible for low plasma BCAA levels. Accordingly, it is recommended that metabolic assessment of patients be performed early after NDD and autism diagnosis and BCAA supplementation be initiated in those with low levels of these amino acids to prevent irreversible brain damage.

Ethical approval

Approval has been given by ethical committee of Shahid Beheshti University of medical Sciences.

Consent

Written informed consent was obtained from the patient’s parents/legal guardian for publication and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request. The patient consent form is the official form Firoozabadi hospital and is available for patient.

Sources of funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Author contributions

All authors have made significant contributions to this case report study. M.R. designed of the work, and wrote the main manuscript text. A.T. supervised the implementation of the plan and edited main manuscript text. M.K. supervised the implementation of the plan and edited main manuscript text.

Conflicts of interest disclosure

The authors declare no conflict of interest in the creation and publication of this article. Our aim is to provide accurate and unbiased information, adhering to strict ethical standards. This work is based on thorough research and analysis, and any potential sources of influence have been fully disclosed. We are committed to maintaining transparency and integrity in all our endeavors, fostering the trust and confidence of our readers.

Research registration unique identifying number (UIN)

This study is not a clinical trial study and it is case report.

Guarantor

Maryam Kachuei.

Provenance and peer review

Not commissioned, externally peer-reviewed.

Data availability statement

All information (without personal information of patient such as name and surname, etc.) is available for review by the Editor-in-Chief of this journal on request.

Presentation

None.

Declarations

We are also thankful to the children and their parents who participated in the present study.