Treatable childhood neuronopathy caused by mutations in riboflavin transporter RFVT2

Abstract

Childhood onset motor neuron diseases or neuronopathies are a clinically heterogeneous group of disorders. A particularly severe subgroup first described in 1894, and subsequently called Brown-Vialetto-Van Laere syndrome, is characterized by progressive pontobulbar palsy, sensorineural hearing loss and respiratory insufficiency. There has been no treatment for this progressive neurodegenerative disorder, which leads to respiratory failure and usually death during childhood. We recently reported the identification of SLC52A2, encoding riboflavin transporter RFVT2, as a new causative gene for Brown-Vialetto-Van Laere syndrome. We used both exome and Sanger sequencing to identify SLC52A2 mutations in patients presenting with cranial neuropathies and sensorimotor neuropathy with or without respiratory insufficiency. We undertook clinical, neurophysiological and biochemical characterization of patients with mutations in SLC52A2, functionally analysed the most prevalent mutations and initiated a regimen of high-dose oral riboflavin. We identified 18 patients from 13 families with compound heterozygous or homozygous mutations in SLC52A2. Affected individuals share a core phenotype of rapidly progressive axonal sensorimotor neuropathy (manifesting with sensory ataxia, severe weakness of the upper limbs and axial muscles with distinctly preserved strength of the lower limbs), hearing loss, optic atrophy and respiratory insufficiency. We demonstrate that SLC52A2 mutations cause reduced riboflavin uptake and reduced riboflavin transporter protein expression, and we report the response to high-dose oral riboflavin therapy in patients with SLC52A2 mutations, including significant and sustained clinical and biochemical improvements in two patients and preliminary clinical response data in 13 patients with associated biochemical improvements in 10 patients. The clinical and biochemical responses of this SLC52A2-specific cohort suggest that riboflavin supplementation can ameliorate the progression of this neurodegenerative condition, particularly when initiated soon after the onset of symptoms.

Keywords: Brown-Vialetto-Van Laere syndrome; RFVT2; SLC52A2; childhood neuronopathy; riboflavin therapy.

Figure 1

Index case from Dr Charles Henry Brown’s original report (Brown, 1894). Reproduced with permission from Kluwer Academic Publishers.

Figure 2

Mutations in SLC52A2 in Brown-Vialetto-Van Laere syndrome. (A) Predicted transmembrane domains in RFVT2, gene structure and location of mutations identified in SLC52A2 in this patient cohort. Reference sequence NM_024531.4. The Washington Exome Variant Server (http://evs.gs.washington.edu/EVS/), single nucleotide polymorphism database (dbSNP) (http://www.ncbi.nlm.nih.gov/snp) and 1000 Genomes Project (http://www.1000genomes.org/) databases were screened for the identified mutations. (B) Structural conservation of relevant amino acid residues in RFVT2 across species and in RFVT1 and RFVT3. Dark blue, medium blue and light blue colours correspond to amino acids conserved in ≥6, ≥5 or ≥3 of 7 sequences, respectively. Conservation among species of the affected amino acid residues was determined using Ensembl to retrieve the sequences and Clustal Omega software (Thompson et al., 1994) for multiple sequence alignment. The Ensembl protein IDs for the RFVT2 orthologous sequences reported are ENSGGOP00000028056, ENSSSCP00000028741, ENSMUSP00000023220 and ENSDARP00000045674.

Figure 3

Phenotypic characteristics of Brown-Vialetto-Van Laere syndrome caused by mutations in SLC52A2. Severe weakness of neck extension and upper limbs with comparatively less weakness of lower limbs seen in Patient I1 at 1.8 years of age (A), Patient E4 at 5.8 years of age (B) and Patient E1 at 8.6 years of age (C). Symmetrical atrophy of intrinsic hand muscles of Patient E1’s left (D) and right (E) hands at 10.5 years of age.

Figure 4

Sural nerve pathology in patients with mutations in SLC52A2. Resin semi-thin sections, stained with methylene blue–azure A and basic fuchsine; ×63 magnification (A and C) and toluidine blue; ×40 magnification (F) and electron microscopy examination (B, D and E) of sural nerve fascicle cross sections in Patient E2 at 2 years of age (A and B), Patient E3 at 4 years of age (C and D), Patient E5 at 4 years of age (E), and Patient A3 at 3 years of age (F) demonstrate a loss of myelinated axons, preferentially of large diameters (8–12 µm). The endoneurium is fibrotic, and there are no inflammatory infiltrates. Numerous redundant Schwannian profiles (Bands of Büngner) are discernible ultrastructurally (B and D, arrows) consistent with the loss of myelinated fibres. Several myelinated fibres in the biopsy of Patient E5 (E) appear vacuolated and slightly enlarged—probably an artefactual change not to be confused with giant axons. Note the striking absence of regeneration clusters in all biopsies. Scale bars: B and D = 5 µm; E = 50 µm.

Figure 5

Functional studies of SLC52A2 mutations. (A) Uptake of ³H-riboflavin by HEK293 cells transfected with empty vector (Vector), wild-type SLC52A2 (WT), SLC52A2 (92G > C; W31S), SLC52A2 (700C > T; Q234X), SLC52A2 (851C > A; A284D), SLC52A2 (914A > G; Y305C), SLC52A2 (916G > A; G306R), SLC52A2 (935T > C; L312P) and SLC52A2 (1016T > C; L339P). The cells were incubated with 5 nM ³H-riboflavin (pH 7.4) for 1 minute at 37°C. Each bar represents the mean ± SEM, n = 3. Data were analysed by Dunnett’s two-tailed test after one-way ANOVA. *P < 0.05, ***P < 0.001, significantly different from vector-transfected cells. ^#P < 0.05, ^###P < 0.001, significantly different from SLC52A2 (WT)-transfected cells. (B) Western blot analysis was performed using the crude membrane of HEK293 cells expressing empty vector, SLC52A2 (WT) and SLC52A2 variants. The crude membrane fractions were subjected to western blotting using antibodies against FLAG and Na⁺/K⁺-ATPase. Na⁺/K⁺-ATPase was used as an internal standard. (C) RNA expression of SLC52A2 in HEK293 cells transfected with empty vector, SLC52A2 (WT) and SLC52A2 variants. Reverse transcription-PCR analysis was carried out using specific primer sets.

Figure 6

Autosomal recessive inheritance of SLC52A2 mutations. Pedigrees and corresponding SLC52A2 mutations for Patients E2, E4, U1, U2, A1, A2, A3, A4, A5, A6 and A7. Squares denote males, circles females, shaded shapes affected individuals, and shapes with dots carriers. Double bars indicate consanguineous unions, and arrows indicate probands.