A wide spectrum of clinical and brain MRI findings in patients with SLC19A3mutations

The experiments were conducted after approval by the institutional review boards at the Institute for Developmental Research, Aichi Human Service Center and Brain Research Institute, Niigata University. Written informed consent was obtained from the parents of the four patients participating in this study.

The proband was an 18-year-old Japanese boy born to healthy consanguineous parents (Figure 1): his maternal grandmother and father are first cousins. Notably, his three cousins (Figure 1 patients V-3, V-4, and V-6) had very similar clinical courses and features. This patient was born at a gestational age of 38 weeks without complications. At 1 month of age, he presented an opisthotonic posture, and at 2.5 months of age, epileptic spasms appeared. His EEG demonstrated multifocal spikes but no hypsarrhythmia; thus, he was diagnosed with atypical infantile spasms without hypsarrhythmia. A complete blood count, biochemical analyses for a wide range of metabolic disorders and an immunohistochemical analysis of skeletal muscle biopsies were normal. Two courses of ACTH therapy were performed but were only transiently effective. At 1 year of age, intractable tonic seizures appeared, and he showed severe mental retardation and spastic quadriplegia with an increased jaw-jerk reflex and hyperreflexia in the extremities, including positive Babinski sign. Involuntary movement was not observed. Brain MRI at 4 months of age revealed symmetrical abnormal intensities in the bilateral thalami and basal ganglia on T1- and T2-weighted images (Figure 2A and 2B). Profound brain atrophy progressed at 1 year (Figure 2C, 2D, 2E and 2F). At 12 years of age, gastrostomy was performed because of swallowing difficulties. At present, he is bedridden with contractures of the extremities and has several tonic seizures daily.

Two other patients who are cousins of patient V-2 and born to parents who are second cousins also had quite similar clinical course and brain MRI findings (Figure 2G, H, I, J, K, L and Table 1). Patients V-3 and V-4 died of respiratory failure at 12 years and 3 months and 9 years and 7 months of age, respectively.

Patient V-6 was born at 40 weeks gestation without complications. He is the fourth child and the younger brother of patients V-3 and V-4. At 11 months of age, he began experiencing epileptic spasms in clusters. His EEG demonstrated anterior dominant irregular diffuse spikes and waves, and polyspikes and waves; however, hypsarrhythmia was not observed. He was diagnosed with atypical infantile spasms. At 1 year of age, he was admitted to our hospital for treatment for epilepsy. He was able to eat weaning food; however, he did not exhibit head control and was unable to roll over. Neurological examination revealed generalized hypotonia, and deep tendon reflexes of the extremities were not elevated. ACTH therapy was transiently effective in alleviating the attacks. Brain MRI at 14 months of age demonstrated severe cortical atrophy and abnormal intensities in the bilateral thalami and basal ganglia. Physical examination at 2 years of age demonstrated severe psychomotor retardation; he was not able to sit without support and was also unable to roll over. Neurological examination demonstrated generalized hypotonia with spasticity of limbs and severe mental retardation. Involuntary movement was not observed. Because he was identified as homozygous for a SLC19A3 mutation, patient V-6 had taken biotin (5 mg/kg/day) for one year beginning at 19 months of age with informed consent from his parents; however, his neurological symptoms and brain MRI findings did not improve. Brain MRI at 3 years and 5 months of age demonstrated diffuse brain atrophy and abnormal intensities in the bilateral thalami, caudate nuclei and putamen on T1- and T2-weighted images (Figure 2M, 2N and 2O). Currently, Patient V-6, at 6 years of age, is bedridden. The clinical features and brain MRI findings of the four patients are summarized in Table 1.

We performed a genome-wide linkage analysis in ten family members related to the four neurological patients using 763 microsatellite markers (Applied Biosystems, Foster City, CA) to cover the all autosomes with an average interval of 4.6 cM. Multi-point LOD (logarithm of odds) scores were calculated using the Allegro program for all loci under the assumption of autosomal recessive inheritance and a disease frequency of 0.001 [13]. Haplotypes were constructed manually to minimize the number of recombination events.

Genomic DNA was isolated from white blood cells by phenol/chloroform extraction. PCR-amplified DNA fragments were isolated with a QIAEX II Gel Extraction Kit (QIAGEN, Valencia, CA) and purified using polyethylene glycol (PEG 6000) precipitation. Isolated PCR products were cloned and sequenced with an SQ5500E DNA sequencer (HITACHI, Tokyo, Japan), or were sequenced directly with specific inner primers or the same primers used for PCR [14].

First-strand cDNA was synthesized by reverse transcription of 10 μg of total RNA from normal or the proband's lymphoblastoid cells using a First-Strand cDNA Synthesis Kit (GE Healthcare, Tokyo, Japan) with specific antisense primer (A1: 5'-GTTGCGTCTAGATTAGAGTTTTGTTGAC-3', XbaI site is underlined) in a reaction volume of 15 μl. The cDNA products were amplified with LA-Taq DNA Polymerase (TAKARA BIO INC, Otsu, Japan) using the specific primers (S1: 5'-AACAGACACTCCCTTCTGAATTCATG-3' and A1, EcoRI site is underlined). PCR was performed in a total volume of 20 μl for 36 cycles as follows: denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 90 s. The 1,535-bp PCR products were subcloned into the EcoRI/XbaI sites of the pCI-neo vector, a mammalian expression vector driven by a cytomegalovirus (CMV) promoter and enhancer, to generate pCI-neo-SLC19A3(WT) and pCI-neo-SLC19A3(E320Q).

We performed a thiamin transport assay according to the protocol of Ashokkumar et al. [15]. Briefly, a 1.0 μg aliquot of the SLC19A3 expression vector (pCI-neo-SLC19A3(WT), pCI-neo-SLC19A3(E320Q)) and the control vector (pCI-neo) were transfected into HEK293 cells in 24-well plates using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA). Two days after transfection, the HEK293 cells were washed two times with pre-warmed phosphate-buffered saline (PBS) and incubated for 10 min at 37°C in 200 μl of assay buffer (20 mM HEPES (pH 7.4), 140 mM NaCl, 5 mM KCl, 2 mM MgCl₂, and 5 mM sucrose) containing 0.1 μM of [³H]-labeled thiamin (14.8 kBq:0.74TBq/mmol) with or without 0.1 mM unlabeled thiamin. Thiamin transport was terminated by the addition of 0.5 ml of ice-cold PBS followed by immediate aspiration. Cells were rinsed two times with 0.5 ml of ice-cold PBS and lysed with 200 μl of 0.2 M NaOH for 30 min at 65°C. A 50-μl aliquot of lysate was spotted onto Whatman 3 MM chromatography paper (GE Healthcare), and radioactivity counts were conducted using a liquid scintillation counter. Protein concentrations were estimated using an Advanced Protein Assay Kit (Cytoskeleton Inc. Denver, CO) according to the manufacturer's instructions.

The patients' characteristic clinical features without obvious extrapyramidal symptoms, rapid clinical course and brain MRI findings did not lead us to diagnosis a specific disease. However, we conducted a genome-wide linkage analysis that revealed a disease locus in the region from D2S2382 to D2S206 on 2q35-37 (~16.7 Mb) with a maximum multipoint LOD score of 4.75 (Figure 1).

SLC19A3 gene maps in the region, and then we identified a homozygous mutation (c.958G > C, [p.E320Q]) in exon 3 of SLC19A3 in all patients, while their parents were heterozygous for the mutation (Figure 3A and 3B).

To address the effect of the E320Q mutation on thiamin transport activity, we transfected wild-type and mutant SLC19A3 expression constructs into HEK293 cells. We then analyzed the uptake of [³H]-labeled thiamin into these cells. We found that the E320Q mutation decreased thiamin uptake to 63% of that of the wild-type protein (Figure 3C), corroborating the findings of Kono et al. [12] using Chinese hamster ovary (CHO) cells.