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Advances in molecular genetic technologies have improved our understanding of genetic causes of rare neurological disorders with features of myoclonus.
A family with two affected siblings, presenting with multifocal polymyoclonus and neurodevelopmental delay, was recruited for whole-exome sequencing following unyielding diagnostic neurometabolic investigations. Compound heterozygous mutations in TBC1D24, a gene previously associated with various epilepsy phenotypes and hearing loss, were identified in both siblings. The mutations included a missense change c.457G>A (p.Glu157Lys), and a novel frameshift mutation c.545del (p.Thr182Serfs*6).
We propose that TBC1D24-related diseases should be in the differential diagnosis for children with polymyoclonus.
Myoclonus is defined as a sudden, brief (less than 100 ms), shock-like muscle contraction involving agonist and antagonist muscles, leading to a sudden jerky movement.1 The lifetime prevalence of myoclonus is estimated to be 8.6 cases per 100,000 population.2 Physiological myoclonus, in the form of hiccoughs or hypnic jerks, is easily recognized. Pathological myoclonus has a wide range of etiologies including acquired/hypoxic neurological injury, as well as neoplastic, infectious, post-infectious, metabolic, and genetic causes. There are a number of ways in which myoclonus is classified, including by anatomical/physiological origin (cortical, subcortical, spinal, peripheral), or by etiology. In the approach to pathological myoclonus, it is useful to consider the distribution of myoclonus, age of onset, insidious/acute onset, triggering/alleviating factors and associated signs or symptoms (Supplementary Table 1).
Recently, advances in molecular genetic technologies have enabled accelerated gene discovery, adding to our understanding of rare neurological disorders, including myoclonus (Supplementary Table 1).
We describe two siblings with infantile-onset multifocal polymyoclonus at rest who were found on whole-exome sequencing to have mutations in TBC1D24, a gene previously associated with various epilepsy phenotypes (including familial infantile myoclonic epilepsy, migrating partial seizures of infancy),3–13 hearing impairment,14–17 and DOORS (deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures) syndrome.18 Prolonged electroencephalograms (EEGs) revealed no epileptiform features; furthermore, multiple EEGs captured episodes of polymyoclonus and demonstrated no EEG correlation.
To our knowledge, this is the first report of TBC1D24 mutations in a phenotype with prolonged, multifocal polymyoclonus as the main presenting feature with no discernible features of epilepsy.
A family with two affected children was ascertained and recruited for molecular genetic analysis. The participating family gave written informed consent and the study was performed in accordance with the Declaration of Helsinki. Genomic DNA from the affected individuals, and both parents was extracted from peripheral lymphocytes by standard techniques.
Whole-exome sequencing was carried out in both affected individuals using Illumina’s TruSeq Exome Enrichment kit (Illumina, Inc San Diego, California, USA), according to manufacturer’s recommendations. Sequencing was performed on Illumina HiSeq2000 using 100-bp paired-end reads. Data were analyzed following Genome Analysis Toolkit’s (GATK) Best Practices (PMIDs, 20644199, 21478889, 25431634). To confirm the identified TBC1D24 variants direct Sanger sequencing was performed. The appropriate exon amplified by polymerase chain reaction (PCR) (primer sequences and PCR conditions on request) was directly sequenced by the Big Dye Terminator Cycle Sequencing System (Applied Biosystems Inc.) on an ABI PRISM 3730 DNA Analyzer (Applied Biosystems Inc.) and analyzed using Chromas (http://www.technelysium.com.au/chromas.html).
We present two siblings born to non-consanguineous parents of Polish descent. There is no significant family history.
The older sibling, A1, was diagnosed with cardiac arrhythmia antenatally, and was born via elective caesarean section at term. At the age of 5 weeks, his mother noted he had intermittent mouth-twitching movements while breastfeeding. This progressed to paroxysmal myoclonic twitching and jerking movements involving varying muscle groups, including those of his eyelids, face, lips, abdomen, and limbs. Myoclonic episodes ranged from short, spontaneously resolving tolerated events with small amplitude twitches to prolonged distressing events with larger amplitude jerks (Video 1A,B). He had preserved awareness with most episodes, but some prolonged episodes resulted in autonomic disturbance such as pallor, sweating, and reduced responsiveness. In casualty, prolonged episodes of myoclonus were often treated as status epilepticus. Benzodiazepines and rectal paraldehyde were reported to terminate some events. Although his symptoms were initially paroxysmal, by the age of 2 years he had almost constant myoclonus involving varying muscle groups. These symptoms were exacerbated by fatigue and abolished by sleep.
At the age of 10 months, A1 received a pacemaker for third-degree atrioventricular block. His development was delayed. He started walking unsteadily at 3 years and said his first word at 3 years of age. At 16 months of age, he was diagnosed with extreme hypermetropia. An early hearing screen was normal, but at the age of 5 years, he developed profound sensorineural hearing loss. On examination, he had dysplastic ears, downslanting palpebral fissures, silvery pigmentation to his hair, and a full philtrum. His weight was on the 25th centile and his head circumference was between the 9th and 25th centile. He had bilateral rotatory nystagmus. He had truncal hypotonia but increased peripheral tone, with a dynamic component, in all four limbs. Deep tendon reflexes were brisk in his lower limbs, with flexor plantar responses bilaterally. Asynchronous migratory myoclonus involving various muscle groups was easily observed throughout the examination.
Patient A2 had an unremarkable perinatal history. She started having myoclonic twitching and jerking in her limbs at the age of 6 weeks. Like her brother, this progressed to paroxysmal episodes of myoclonus, most often involving the abdomen or face. As she grew older, her myoclonus also became more widespread and constant.
A2 did not have cardiac symptoms. There have been no concerns regarding her vision or hearing. Currently, at the age of 3 years, she has poor balance and coordination in addition to speech delay. She does not have abnormal eye movements. She has normal tone and deep tendon reflexes.
Extensive neurometabolic investigations performed on blood, cerebrospinal fluid (CSF) and urine for both siblings revealed nothing of note apart from a mildly reduced CSF glucose on one occasion for A1 (1.9 mmol/l, plasma glucose 4.4 mmol/l) and two occasions for A2 (1.7 mmol/l, plasma 4.5 mmol/l; and 1.9 mmol/l, plasma 4.7 mmol/l). For both individuals, these normalized on repeat testing.
A1 had a normal male karyotype and normal microarray results. Testing for mutations in candidate genes for GLUT1 deficiency syndrome and mitochondrial disorders, including SLC2A1, POLG, and DGOUK, yielded negative results.
Repeated EEGs including prolonged EEGs encompassing sleep for both siblings showed a mild excess of slow activity, but no evidence of epileptiform activity. Polygraphic electromyogram (EMG) recordings of right and left deltoids, biceps, forearm, and quadriceps muscles demonstrated myoclonias of approximately 80–110 ms duration in all the EMG channels without any evidence of spread from one to another. A few myoclonias were more hypersynchronous, approximately 40–60 ms in duration (Supplementary Figure 2). Back-averaging did not reveal any preceding EEG change in either child. Nerve conduction studies and somatosensory evoked potential tests were normal.
A1 had serial cranial magnetic resonance imaging (MRI) scans from the age of 2 months to 19 months. These demonstrated hypoplasia of the frontal and temporal lobes. The deep grey matter appeared normal with no evidence of cortical dysplasia (Figure 1A). A computed tomography (CT) head scan showed tiny non-specific foci of calcification in the basal ganglia (Figure 1B).
Cranial MRI and MR spectroscopy performed at 6 weeks of age for A2 were normal. However, a repeat MRI scan at 3 years 10 months showed atrophy of the lateral aspects of the cerebellar hemispheres and symmetrical signaling abnormalities (Figure 1C).
A1 received trials of treatment with an extensive range of medication including anti-epileptics (valproate, carbamazepine, nitrazepam, levetiracetam, clobazam), metabolic supplements (coenzyme Q10, biotin, riboflavin, thiamine), levodopa, and piracetam. None of these achieved sustained benefit, although levetiracetam and clobazam each controlled myoclonus briefly. Immunotherapy had no significant effect.
Levetiracetam exacerbated A2’s symptoms and pyridoxine was ineffective. She did not tolerate the ketogenic diet. Bromocriptine resulted in dyskinetic movements, which terminated with discontinuation of the medication. Carbamazepine afforded her some symptomatic relief.
For both siblings as symptoms were abolished by sleep, the best therapeutic strategy seemed to be chloral hydrate used, as required, to induce sleep during periods of distress.
Variants identified through whole-exome sequencing were filtered by the following criteria: 1) very low frequency in control populations (exclusion of variants with a minor allele frequency > 0.1% in the established databases and 2,000 in-house analyzed exomes); 2) presentation of homozygous or compound heterozygous changes considering an autosomal recessive inheritance pattern; and 3) prediction of putative pathogenicity based on mutation type or in silico prediction of effects on protein function and/or structure. Using these criteria, two heterozygous variants confirmed by Sanger sequencing were identified in TBC1D24 (Supplementary Figure 1): 1) a previously reported missense change c.457G>A (p.Glu153Lys; rs376712059)6,13,15 and 2) a previously unreported frameshift mutation, c.545del (p.Thr182Serfs*6) predicted to cause nonsense-mediated RNA decay or result in a truncated protein. Both mutations showed appropriate familial segregation.
Other possible disease-causing mutations identified in the two subjects that passed selection criteria were excluded (Supplementary Table 2). Mutations found in two genes (NOTCH4 and PRR21) could not be verified as it was not possible to design primers for confirmatory Sanger sequencing and segregation studies because of highly repetitive sequences. We note that whole-exome sequencing false-positive rates for indels can be as high as nearly 50%.19 Considering their phenotype, TBC1D24 was thus deemed to be the most likely candidate gene accounting for our patients’ symptoms.
Myoclonus is rare and confirmation of a definitive diagnosis can often prove challenging. A definitive genetic diagnosis facilitates pre-pregnancy counseling and ends an often long diagnostic odyssey. Identification of causative genes can also contribute to the understanding of disease mechanisms and may facilitate development of novel targeted therapies. However, obtaining a genetic diagnosis is often complicated by genetic heterogeneity and phenotypic pleiotropy. Our report further illustrates how molecular genetic advances can facilitate gene discovery and clinical diagnosis.
TBC1D24 (OMIM 613577) encodes Tre2/Bub2/Cdc16 (TBC) 1 domain family member 24, a member of a family of Rab-specific GTPase-activating proteins.12 These have a role in coordinating Rab proteins and other GTPases for transport of intracellular vesicles. TBC1D24 interacts with ADP ribosylation factor 6 (ARF6), a GTPase with an essential role in membrane trafficking.5,20 The Rab GTPases control neuronal cell morphology and migration. In addition, TBC1D24 has a TBC lysine motif catalytic (TLDc) domain, thought to have a role in oxidative stress resistance.18,21 Loss of TBC1D24 function may result in abnormal vesicle trafficking, abnormal neuronal migration/maturation, and neurodegeneration.12,18,21
Mutations in TBC1D24 have been described in an array of disorders summarized in Table 1. Overall, among disorders associated with TBC1D24 mutations, recurring phenotypes include seizures, myoclonus, neurodevelopmental impairment, sensorineural hearing impairment, visual impairment, and cerebral and/or cerebellar atrophy on brain imaging. Movement disorders including dystonia, choreoathetosis, and dyskinesia have also been reported.12,13 Our sibship shares a number of clinical features with previously reported TBC1D24 cases. These include myoclonus, cerebellar atrophy, and neurodevelopmental impairment. A1 also had visual impairment and developed sensorineural hearing impairment.
To our knowledge, TBC1D24 mutations have not been reported in a phenotype involving prolonged, almost continuous multifocal myoclonus as the main presenting feature with no discernible epileptiform features on repeated interictal and ictal EEG recordings. Doummar et al.22 reported a case with similar multifocal myoclonus and homozygous c.809G>A (p.Arg270His) TBC1D24 mutations. However, unlike our sibship, interictal EEGs of this case presented paroxysmal epileptiform abnormalities and there was evidence suggesting his myoclonus had cortical origin. The myoclonia our sibship present with are more variable, asynchronous, and isolated, with a multifocal segmental pattern, than Doummar’s case, who had synchronous, generalized bursts of myoclonia. Interestingly, both siblings also had frequent abdominal myoclonus, which is rare and associated with myoclonus of spinal origin.
In conclusion, our report expands the TBC1D24 mutation spectrum by describing a novel mutation and extends the gene’s phenotypic spectrum. It suggests TBC1D24 should be considered amongst candidate genes in children with myoclonus and neurodevelopmental impairment even in the absence of clear epileptiform features.
All supplementary data referenced in this article is available here: http://dx.doi.org/10.7916/D8QN6CZG.
Funding: A.N. is funded by a Guarantors of Brain Entry Fellowship and an Action Medical Research Training Fellowship. J.B. and R.G.’s work is supported by Fellowships from the Alzheimer’s Society. A.M. and J.N. are funded by Medical Research Council Clinical Research Training Fellowships. A.M. also receives funding from Medical Research Foundation, Child Brain Research and Young Epilepsy; and J.N. is also supported by Great Ormond Street Hospital Children’s Charities. M.A.K. is funded by a Wellcome Trust Intermediate Clinical Fellowship and receives funding from the Rosetrees Trust, Gracious Heart Charity Foundation Great Ormond Street Hospital Children’s Charities (GOSHCC) Child Brain Research and Rachel Marie Trafford Trust.
Financial Disclosures: None.
Conflict of Interest: The authors report no conflict of interest.
Ethics Statement: All patients that appear on video have provided written informed consent; authorization for the videotaping and for publication of the videotape was provided.