Background Recessive TBC1D24 gene mutations have been described in two families: an Italian family afflicted with familial infantile myoclonic epilepsy, and an Arab family with focal epilepsy and intellectual disability syndrome. The patients in the Italian family were compound heterozygous for two mutations, whereas those in the Arab family were homozygotes. All three mutations were missense and were determined to be loss of function. We conducted a gene search in a family we previously reported with a severe, lethal epileptic encephalopathy mapping at 16pter-p13.3.
Methods Exome sequencing and subsequent Sanger sequencing of TBC1D24 exons were conducted. Sanger sequencing was used to determine the structures of novel mRNA isoforms. The abundance of mRNA isoforms was assessed via real-time quantitative PCR.
Results A homozygous two-base pair deletion leading to premature termination and two novel TBC1D24 transcript isoforms were identified. Isoform 1 is predominant in the brain whereas isoform 2 is predominant in non-neural tissues, except for muscle.
Conclusions The very severe phenotype in our patients can be attributed to mutation severity; however, the mutation does not affect isoform 2, whereas the three previously reported mutations do. These findings expand the spectrum of the TBC1D24 mutation phenotype and the transcript isoforms.
- Epilepsy and seizures
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TBC1D24 (TBC1 domain family, member 24; MIM 613577) was originally isolated from a brain cDNA library.1 The deduced TBC domain suggested that the protein was involved in cell signalling. In 2010, Falace et al reported that TBC1D24 binds to ARF6 (ADP-ribosylation factor 6) and suggested that TBC1D24 was a negative regulator of the function of ARF6, which is known to regulate axonal elongation and branching.2 ,3 Recessive TBC1D24 mutations were reported in two families afflicted with different diseases.3 ,4 An Italian family was afflicted with familial infantile myoclonic epilepsy (FIME; MIM 605021) characterised by myoclonic and generalised tonic-clonic seizures, photosensitivity, and normal neurological and mental development.3 ,5 ,6 An Arab family had focal epilepsy and intellectual disability syndrome associated with subtle cortical thickening that was most obvious in the anteromesial frontal areas. In addition to convulsive seizures, the disease was characterised by focal seizures with prominent eye blinking and facial and limb jerking that began in infancy and persisted throughout life; in adulthood there was borderline to moderate intellectual disability associated with mild dysarthria and ataxia.4 The Italian patients were compound heterozygous carriers for two missense mutations, whereas the Arab patients were homozygous for another missense mutation. Patients in both families responded positively to antiepileptic medication.
We previously mapped to 16pter-p13.3 a severe disease characterised by early onset progressive encephalopathy associated with myoclonic epilepsy with dystonia, and developmental and neurological retardation that led to full deterioration and death during the first decade of life.7 The patients did not respond to medication. Additional manifestations of the disease were episodic phenomena, including dystonias, postictal enduring hemipareses, autonomic involvement, and periods of obtundation and lethargy. A patient that was given an ocular examination during late-stage disease had bilateral optic atrophy, macular degeneration, dilated pupils, and no light response. MRI findings in another patient were diffuse moderate cerebral atrophy and ventricular enlargement at 14 months of age, and severe diffuse cerebral atrophy with secondary ventricular enlargement at 37 months, indicating significant progression of the encephalopathy.7 In contrast, MRI findings in the Italian patients were normal.5 The findings in the Arab patient that was investigated were interpreted as thickened cortex in the frontal poles with loss of grey-white matter definition that was consistent with a developmental malformation.4 Thus, although the disease began in the first year of life with myoclonic seizures in all three families, it evolved differently in each.
Herein we report a homozygous truncating TBC1D24 mutation in the recently identified alternative exon in our patients. Additionally, we compared the abundance of the isoforms in various human brain regions and non-neural tissues.
Subjects and methods
In all, five affected individuals were known in the consanguineous family. We had performed genetic analysis in 26 family members to map the disease locus.7 Informed consent was obtained from/for all participants, and the study protocol was approved by the Boğaziçi University Institutional Review Board for Research with Human Participants.
DNA was isolated from peripheral blood samples. A good candidate mutation was not found in a patient sample via exome sequencing analysis using the Roche Nimblegen V.1.0 Exome Array (see online supplementary table S1). Concurrently, TBC1D24 mutations in two other families were independently reported.3 ,4 As this gene was not covered by our exome sequencing analysis, we sequenced all eight of its exons and flanking sequences in a patient. In order to screen the family members and population controls for the identified mutation, a 237 bp region around the mutation was amplified via PCR. The alleles were resolved on single-strand conformation polymorphism (SSCP) gels and visualised following silver nitrate staining. The sequences of all primers used in this study are shown in online supplementary table S2.
During the first attempt to investigate the abundance of TBC1D24 transcript isoforms, a primer specific to the junction of exons 2 and 3 in transcript isoform 1, and another primer specific to the junction of exons 2 and 4 in transcript isoform 2, were utilised together with a common reverse primer (see online supplementary table S2). As the quantitative real-time PCR products were analysed on agarose gels for size and purity, two aberrant fragments were observed in the isoform 1 amplification samples. The fragments were purified using a QIAQuick Gel Extraction Kit (Qiagen) and subjected to sequence analysis. One fragment was designated as isoform 3 and the other as isoform 4. The structure of each isoform was investigated further via sequencing following amplification using a forward primer specific to the isoform and a reverse primer specific to exon 8 (see online supplementary table S2).
Quantification of transcript isoforms
To investigate the abundance of TBC1D24 transcript isoforms in seven adult human brain regions, we attempted to amplify each isoform separately in an isoform-specific quantitative PCR assay; however, isoform 1 could not be amplified alone, as it contained all of the exons known to us. A forward primer specific to the junction of exons 3 and 4 was used to amplify isoform 1 together with isoform 4, a primer specific to the junction of exons 2 and 4 was used to amplify isoform 2, and a primer specific to the junction of exons 3 and 5 was used to amplify isoform 3, together with a common reverse primer specific to exon 6 (see online supplementary table S2). Total RNA samples (Clontech Laboratories) were used as templates to synthesise cDNA using a RevertAid First Strand cDNA Synthesis Kit (Fermentas). Real-time PCR was performed using an SYBR Green I Master Kit on a LightCycler 480 (Roche). CT values for the reactions were obtained using LightCycler 480 V.1.5 software, and relative quantification was performed via the 2(-Delta C(T)) method.8 All reactions were normalised to endogenous reference gene HPRT1, and calculations were performed in Microsoft Excel. Each assay was performed in triplicate. A similar assay was performed to assess mRNA abundance in five non-neural tissues using a primer specific to the junction of exons 2 and 4 in isoform 2 and a primer specific to exons 2 and 3 in isoforms 1, 3 and 4, together with a reverse primer specific to exon 6 (see online supplementary table S2).
First, the seven exons of TBC1D24 that were known at the time were analysed by DNA sequencing in one patient, but no mutation was observed. Then, the 18 bp exon 3 (NM_001199107.1), which was newly reported, was sequenced, and the deletion of one of the three tandem GT dinucleotides was identified (see online supplementary figure S1A). This 2 bp deletion results in a shift in the translational reading frame and causes a premature termination at codon 326, following the synthesis of two non-native amino acids (NM_001199107.1: g.1109_1110delGT, NM_001199107.1: c.969_970delGT and NP_001186036.1: p.Ser324Thrfs*3). The mutation segregated with the trait in the family and was not observed in a panel of 120 population control samples that was screened, showing with >80% power that the mutation was not a normal sequence variant in the Turkish population.9
In all, two aberrant products, one primarily in the cerebellum sample and one in the striatum sample, were observed during the initial attempt to specifically amplify isoform 1 in brain tissue via quantitative PCR assay (see online supplementary figure S2). Both products were observed in trace amounts in comparison to the main product. Sequence analysis showed that they were specific to novel transcript isoforms (see online supplementary figure S1B,C). The smaller isoform lacked exon 4, whereas the larger isoform lacked exon 5; we designated them as isoform 3 and isoform 4, respectively. We sequenced them further until 3′UTR to ascertain that they included all exons downstream of exon 3. The exonic structures of the isoforms, the deduced structures of the proteins encoded, and the predicted effects of the identified mutation on the protein isoforms are given in figure 1. Transcript isoform 2 is deduced not to be affected by the mutation.
The abundance of transcript isoform 1 (together with isoform 4), isoform 2 and isoform 3 was compared in different human brain regions. All isoforms were observed in all brain regions investigated, namely, the cerebellum, corpus callosum, frontal cortex, occipital cortex, striatum, parietal cortex, and brain stem. In all samples isoform 1 was more abundant than the other isoforms (figure 2A). The abundance of isoforms 1, 3 and 4 (all harbouring exon 3) together and that of isoform 2 alone was investigated similarly in five non-neural tissues, namely, blood (leucocytes), adipose tissue, bone marrow, skeletal muscle and liver. Isoform 2 was much more abundant than the total of all the others in the liver, blood, adipose tissue and bone marrow, whereas in skeletal muscle it was less abundant (figure 2B).
To date, recessive TBC1D24 mutations have been reported in two families: an Italian family afflicted with a form of benign epilepsy, and an Arab family with focal epilepsy and intellectual disability syndrome.3 ,4 Most of the patients in those families were adults whereas our patients with TBC1D24 mutation never walked or talked, had deteriorated severely by the age of 2 years, and died before the age of 8 years. We attributed the greater disease severity in our study family to mutation severity. The mutation is truncating, whereas the mutations in the other two families are missense. The severity of those missense mutations was investigated via their effects on the length and number of neurite termini in primary neurons prepared from mouse embryo brains.3 ,4 The assays for the mutations in the Italian family showed that p.Ala509Val (NP_065756.1) in exon 8 resulted in complete loss of function, whereas p.Asp147His (NP_065756.1) in exon 2 resulted in partial loss of function.3 The patients in the Arab family were homozygous for p.Phe251Leu (NP_065756.1) in exon 2, and the mutation was observed to lead to loss of function.4 Although such an in vitro assay provides information about the function of the gene, it is not an absolute measure of protein activity. Thus, it is possible that some TBC1D24 protein activity has persisted in the Arab patients as well. The mutation in our patients is deduced to lead to a protein 42% shorter than the native protein encoded by isoform 1. The severity of the mutations paralleled disease severity: the phenotype in the Italian patients was benign, that in the Arab patients was moderate, and that in our patients was severe, with rapid progression and early death. Such genotype–phenotype correlation is common in genetic diseases; what is perplexing is that the mutation in our patients is deduced not to have any effect on isoform 2.
TBC1D24 expression is highest in the brain.3 We observed that isoform 1 was the predominant brain isoform whereas isoform 2 was predominant in the non-neural tissues assayed, except for skeletal muscle. The protein products of isoforms 1, 3 and 4, that were deduced to be relatively more abundant in the brain and to harbour exon 3, are possibly subject to regulation, as three of the six amino acids encoded by exon 3 are serine. Serine is the most frequently phosphorylated amino acid, and protein phosphorylation is a mechanism of protein activity regulation.
In conclusion, we believe that the very severe phenotype in our patients was due to mutation severity, even though the mutation does not affect isoform 2. It is now clear that TBC1D24 is not a simple gene with a single function and that it is responsible for a variety of neurological diseases with varying severity. The three families with TBC1D24 mutation reported to date exhibited different clinical phenotypes, except for myoclonic seizures that began during the first year of life, which was common to all three families. We recommend testing all infants with myoclonic seizures of unknown aetiology for TBC1D24 mutations.
We thank the family for participating in this study. Ayse Guven received a fellowship from the Scientific and Technological Research Council of Turkey (110T252). This study was supported by the Boğaziçi University Research Fund (BAP 5708).
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▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/jmedgenet-2012-101313).
Contributors AG generated and analysed data and co-wrote the manuscript; AT supervised genetic analyses and co-wrote the manuscript.
Funding This study was supported by Boğaziçi University Research Fund, grant number BAP 5708.
Competing interests None.
Patient consent Obtained.
Ethics approval The Boğaziçi University Institutional Review Board for Research with Human Participants approved the study protocol.
Provenance and peer review Not commissioned; externally peer reviewed.
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