Myopathic changes associated with psychomotor delay and seizures caused by a novel homozygous mutation in TBCK

Abstract

Introduction:

Biallelic mutations in TBC1-domain containing kinase (TBCK) lead to hypotonia, global developmental delay with severe cognitive and motor deficits, and variable presentation of dysmorphic facial features and brain malformations. It remains unclear whether hypotonia in these individuals is purely neurogenic, or also caused by progressive muscle disease.

Methods:

Whole exome sequencing was performed on a family diagnosed with non-specific myopathic changes via histological analysis and immunohistochemistry of muscle biopsy samples.

Results:

A novel homozygous truncation in TBCK was found in two sisters diagnosed with muscle disease and severe psychomotor delay. TBCK is completely absent in these patients.

Discussion:

Our findings identify a novel early truncating variant in TBCK associated with a severe presentation and add muscle disease to the variability of phenotypes associated with TBCK mutations. Inconsistent genotype/phenotype correlation could be ascribed to the multiple roles of TBCK in intracellular signaling and endolysosomal function in different tissues.

INTRODUCTION

Biallelic mutations in TBC1-domain containing kinase (TBCK, MIM:616899) lead to infantile hypotonia with psychomotor retardation and characteristic facies 3 (IHPRF3, MIM:616900)^1,2. This rare genetic disorder presents with hypotonia, areflexia, epilepsy and severe global developmental delay with variable dysmorphic facial features^2–5. The most severe cases also display variable brain malformations including enlarged ventricles and leukodystrophy, which have been termed TBCK encephaloneuropathy (TBCKE)^3,4. A progressive neuromuscular disease was suspected in some cases. Muscle atrophy was observed via ultrasound in one patient⁴ and “small muscle fibers” were described in electron microscopy in a separate report⁶. Muscle biopsies performed in infancy in a subset of patients were normal or non-diagnostic^3,4,6,7.

Here, we report two sisters with a novel truncating mutation in TBCK who were originally diagnosed with myopathy and neurocognitive delay. Both showed a presentation consistent with TBCKE. Muscle biopsy results show that muscle disease may be present early in life and that neuromuscular involvement can be an aspect of TBCK-related disease.

METHODS

Subjects

The family was enrolled through informed consent with the approval of the Institutional Review Boards of the Fondazione IRCCS Istituto Neurologico Carlo Besta, Rutgers University, and the George Washington University.

Muscle biopsy analysis

Muscle biopsies were flash frozen and cryosectioned for histological examination and immunohistochemistry. Samples were stained with hematoxylin and eosin (H&E), Gomori trichrome, Oil red O, Periodic Acid Schiff, and for nicotinamide adenine dinucleotide (NADH), succinic dehydrogenase, acid and alkaline phosphatase, phosphorylase, non-specific esterase, α-, β-, γ-, δ-sarcoglycan, laminin-α2 N-terminus and C-terminus and α-dystroglycan glycosylation clones VIA4–1, and IIH6 (Suppl. Table 1).

Whole exome sequencing analysis

Whole exome sequencing was performed on deoxyribonucleic acid obtained from the parents and the two affected siblings at the Broad Institute Genomic Services (Broad Institute, Cambridge, MA) using Illumina Nextera Exome kits. Reads were aligned and variants were called and annotated using standard tools^8–11. Variants were filtered for frequency lower than 0.5% in the Genome Aggregation Database¹² and in the Northwest African subgroup of the Greater Middle East Variome Project¹³, for coding changes likely disrupting protein sequence, as well as shared inheritance across the two affected siblings. Heterozygosity in the parents was confirmed by Sanger sequencing at Genewiz (South Plainfield, NJ) and sequencing primers are available upon request.

TBCK expression analysis

Protein lysates were obtained from differentiated myotubes of patients and controls using RIPA buffer and protein extracts were processed for Western blot analysis. Membranes was incubated with primary antibodies raised against TBCK and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Suppl. Table 1) and then with biotinylated goat anti-mouse IgG and peroxidase-conjugated streptavidin (Jackson ImmunoResearch) for chemiluminescent detection (Thermo Fisher Scientific). For immunoflurescence, cryosections from muscle biopsies of both patients were incubated in TBCK primary antibody, and in Alexa 546-conjugated goat anti-mouse IgG (Life Technologies, Thermo Fisher Scientific Inc. Rockford, IL, USA); nuclei were stained with TO-PRO-3 (Thermo Fisher Scientific). Immunostaining was imaged under a confocal microscope (Leica Microsystems).

RESULTS

Case reports

The proband (P1) is a 13-year-old girl and was the first born from Moroccan parents who reported no consanguinity (Table). Pregnancy and delivery were uneventful. During the first month of life, she presented with sucking difficulties and generalized hypotonia. Neurological development was delayed with the achievement of head control at 12 months. At 16 months of age she suffered her first epileptic seizure and neurological examination revealed macrocephaly with occipitofrontal circumference of 48.8 cm (98%), marked generalized hypotonia and weakness, absence of trunk control, areflexia, and intellectual disability with absence of language (Griffith Scale Quotient= 37.7). Dysmorphic facial features are reported in the Table. Electroencephalography showed spikes in the left frontal region. Brain magnetic resonance imaging (MRI) at 4 years of age showed a mild enlargement of lateral ventricles, thin corpus callosum, an enlarged cisterna magna (Suppl. Fig. 1A) and a reduction of hemispheric white matter with a slight T2 hyper-intensity (Suppl. Fig. 1B–C).

Table.

Clinical presentation of the affected individuals

Individual	P1	P2	Published cases1–7
Mutation	c.535_554del; p.Leu179fs	c.535_554del; p.Leu179fs
Sex	Female	Female	34(20M/14F)
Age to date (years)	13	8
Ethnicity	Moroccan	Moroccan
Dysmorphic features	tented upper lip, open mouth, high arched palate, bulged heel	tented upper lip, open mouth, high arched palate, bulged heel	68%
Occipitofrontal circumference	48.8cm (98%)	54cm (99%)
Developmental disability
Cognitive impairment	severe delay	severe delay	100%
Language delay	absent	absent	100%
Regression	no	no	48%
Seizures	focal onset with secondary generalization	focal onset with secondary generalization	71%
MRI findings
Thin corpus callosum	yes	yes	37%
White matter abnormalities	yes	yes	37%
Atrophy	no	no	33%
Enlarged ventricles	yes	yes	21%
Motor function
Motor impairment	motor delay	motor delay	100%
Hypotonia	yes	yes	100%
Reduced/absent reflexes	yes	yes	90%
Weakness	axial and limb weakness	axial and limb weakness
Neuropathy	no	no	In ref [4]
Creatine Kinase	235 UI/I (normal: 24–150)	1,333 UI/I (birth) 141 UI/I (11mo)
Muscle biopsy	myopathic features	small fibers
Miscellaneous	scoliosis, hypertrichosis

Abbreviations: M=males; F=females; mo=months

Creatine kinase (CK) levels were slightly elevated (Table). At the last clinical evaluation at age 12 years she was unable to stand or walk. Seizures were well controlled with valproic acid treatment. Other observations are reported in the Table.

A younger sister (P2) presented with the same clinical features as P1 (Table) though high CK levels at birth normalized by 11 months of age. Epilepsy had been present since 9 months of age and was almost completely controlled with valproic acid treatment.

Neuromuscular assessment

Needle electromyography and nerve conduction studies (NCS) performed in proximal and distal muscles of upper and lower limbs in P1 were normal (Suppl. Table 2). Only NCS were performed on P2 and they were normal. Since muscle disease was suspected, a quadriceps muscle biopsy was performed on both sisters. Muscle from P1 at 18 months of age showed non-specific myopathic changes with marked fiber size variability, a few degenerating or regenerating fibers, and a few fibers (5–6%) with central nuclei seen on H&E staining (Fig. 1A–B) and Gomori trichrome staining (Fig. 1D–F). Muscle biopsy from P2 at 4 years 3 months of age showed mild fiber size variability, a few central nuclei and atrophic fibers, but no degeneration (Fig. 1C and G). Structural fiber changes characteristic of congenital myopathy were not present (NADH staining shown in Fig. 1H–J). Immunohistochemistry showed normal sarcoglycan (not shown) and laminin-α2 expression in both patients (Suppl. Fig. 2A–C), but mildly reduced α-dystroglycan glycosylation (Fig. 1K–M for IIH6 clone and Suppl. Fig. 2D–F for VIA-4 clone).

Figure 1. Neurological and neuromuscular presentation.

A–G. H&E (A–C) and Gomori trichrome (D–G) staining of muscle tissue showed fiber size variability, central nuclei (asterisks in D) and basophilic regenerating fibers (arrows in B, E–F) in P1 compared to a control (Cont) muscle biopsy (A). P2 also showed few central nuclei (asterisk in C) and mild fiber size variability with a few small fibers (arrowhead in C and G). H–J. Nicotinamide adenine dinucleotide (NADH) staining for P1 (I) and P2 (J) compared to an age matched control (H) shows normal distribution. K–M. Reduced dystroglycan glycosylation was found in both P1 and P2 using the IIH6 monoclonal antibody. All scale bars: 50μm

Whole Exome Sequencing and Molecular Analyses

Whole exome sequencing was performed on both sisters. Only one variant was left after analysis, a homozygous 20bp deletion in TBCK [NG_034057 (NM_001163435): c.535_554del; NP_001156907.1:p.Leu179ArgfsTer10] (Fig. 2A). Sanger sequencing validation confirmed inheritance. The deletion is predicted to cause an early truncation in the 893 amino acid protein (Fig. 2B). TBCK expression analysis was performed by Western blot on protein lysates of myoblasts and myotubes (after 10 days differentiation) from patients and controls showing almost complete protein loss in both type of cells (myotubes shown in Fig.2C). Analysis of TBCK expression by immunohistochemistry in control muscle biopsies showed a positive signal localized on muscle fibers often beneath or near the nuclei (Fig. 2D). The TBCK signal was not detected in the muscle biopsies of the patients (Fig.2D).

Figure 2. A novel small deletion leading to TBCK loss-of-function.

A. A homozygous 20bp deletion in TBCK was identified via exome sequencing [NG_034057 (NM_001163435): c.535_554del; NP_001156907.1:p.Leu179ArgfsTer10]. Sanger sequencing was performed to validate the deletion in both parents and affected individuals. B. Protein schematic showing the novel variant and known nonsense, frameshift and missense variants listed above the protein, while splicing variants are reported below. The novel variant identified here is underlined. All variants refer to transcript NM_001163435, variants labeled in blue had previously been reported on transcript NM_133115. C. Western blot of protein lysates generated from myotubes collected from both sisters and controls confirming loss of the TBCK protein in the patients. D. In control muscle (Cont) TBCK is localized on the surface of myofibers and often underneath the nuclei (stained with the TO-PRO-3 dye). No TBCK staining is found in the muscle biopsy from P1 (lower panels). Scale bar: 50μm

Discussion

TBCK mutations lead to severe cognitive and motor delay, but the clinical presentation can vary widely. A portion of these cases show distinctive radiological features on MRI warranting the classification of TBCKE^5,6. Neuronal inclusions in the cerebral cortex and spinal cord reminiscent of a lysosomal storage disorder, both with and without neurodegeneration, have also been reported^4,14–16. It remained unclear whether myopathy was also involved in the hypotonia and weakness observed in the patients. Ortiz-Gonzalez et al suggested progressive muscle loss via ultrasound, which was not detected by biopsies performed in infancy⁴.

We identified a novel 20-bp deletion in TBCK in two individuals with moderate TBCKE. During neurological analysis, non-specific myopathic changes were identified in P1 via muscle biopsy, with a less severe presentation in P2, indicating that muscle disease may also be part of TBCKE progression in some cases. This substantial variability in phenotypes across individuals^2–4 could be ascribed to a pleiotropic effect of TBCK loss of function. TBCK has been shown to regulate the expression of multiple components of the mammalian target of rapamycin complexes (mTORC1 and 2) likely to affect both complexes with widespread effects on cell survival, proliferation and cytoskeletal regulation^17,18. Loss of TBCK leads to decreased mTOR signaling, increased autophagy, accumulation of glycoproteins and decreased mitochondrial function, which can contribute to both developmental and degenerative features of this disorder^2–4,18. mTOR activity is also critical for myocyte differentiation and myotube fusion and maturation¹⁹. While mTORC1 signaling activity is often increased in dystrophic muscle^20,21, mTOR has a kinase-independent role on myotube maturation which could lead to the smaller fiber size observed in our patients and in Mandel et al⁶. While complete ablation of mTOR in skeletal muscle leads to severe myopathy with a reduction in expression of dystrophin, dystroglycan and β-sarcoglycan²², the reduction in dystroglycan glycosylation in these patients is likely non-specific. While limited information on TBCK subcellular localization is available, previous reports had shown perinuclear colocalization with late endolysosomal compartments in cell lines¹⁸. It is possible that reduced surface expression of dystroglycan is caused by disrupted endolysosomal trafficking leading to fewer glycoproteins being delivered to the plasma membrane.

In summary, this study identifies a novel null truncation in TBCK that leads to a phenotype consistent with IHPRF3 and TBCKE including non-specific myopathic changes.

Abbreviations

CK

creatine kinase

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

H&E

hematoxylin and eosin

IHPRF3

infantile hypotonia with psychomotor retardation and characteristic facies 3

IRCCS

Istituto di Ricovero e Cura a Carattere Scientifico

IU/l

International Unit/liter

MRI

magnetic resonance imaging

mTORC

mammalian target of rapamycin complex

NADH

nicotinamide adenine dinucleotide

NCS

nerve conduction studies

TBCK

TBC1-domain containing kinase

TBCKE

TBCK encephaloneuropathy