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) 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%. Considering their phenotype, TBC1D24 was thus deemed to be the most likely candidate gene accounting for our patients' symptoms.