Vitamin B1 deficiency leads to high oxidative stress and mtDNA depletion caused by SLC19A3 mutation in consanguineous family with Leigh syndrome

Leigh syndrome (LS) and Leigh-like spectrum are the most common infantile mitochondrial disorders characterized by heterogeneous neurologic and metabolic manifestations. Pathogenic variants in SLC carriers are frequently reported in LS given their important role in transporting various solutes across the blood–brain barrier. SLC19A3 (THTR2) is one of these carriers transporting vitamin-B1 (vitB1, thiamine) into the cell. Targeted NGS of nuclear genes involved in mitochondrial diseases was performed in a patient belonging to a consanguineous Tunisian family with LS and revealed a homozygous c.1264 A > G (p.T422A) variant in SLC19A3. Molecular docking revealed that the p.T422A aa change is located at a key position interacting with vitB1 and causes conformational changes compromising vitB1 import. We further disclosed decreased plasma antioxidant activities of CAT, SOD and GSH enzymes, and a 42% decrease of the mtDNA copy number in patient blood. Altogether, our results disclose that the c.1264 A > G (p.T422A) variant in SLC19A3 affects vitB1 transport, induces a mtDNA depletion and reduces the expression level of oxidative stress enzymes, altogether contributing to the LS phenotype of the patient.


Introduction
Leigh syndrome (LS) was described for the first time in 1951 by Denis Leigh as a Subacute Necrotizing Encephalomyopathy (Leigh 1951), and is defined today as a complex and incurable early onset pediatric mitochondrial disease which englobes several neurological manifestations (Stendel et al. 2020;Bakare et al. 2021).The common clinical features involve ataxia, hypotonia, developmental delay, seizures associated with dysphagia, failure to thrive, persistent vomiting, elevated serum or cerebrospinal fluid lactate levels, and abnormal ocular disturbances (Gerards et al. 2016;Chang et al. 2020;Ogawa et al., 2020;Stendel et al. 2020).
LS is genetically heterogeneous, resulting in a diverse mode inheritance including maternal or autosomal recessive transmissions, very rare X-linked and autosomal dominant inheritances, as well as de novo variants (Ruhoy and Saneto 2014;Lake et al. 2016;Bakare et al. 2021).Many years after LS first appeared, it was discovered that some patients had deficiency of pyruvate decarboxylase or pyruvate dehydrogenase complex (PDHc) (Evans 1981;Devivo et al. 1979), and altered metabolism of thiamine (Worsley et al. 1965;Baertling et al. 2014;Maas et al. 2017).Thiamine or vitamin B1 (vitB1) is a water-soluble vitamin acting as a cofactor involved in energy metabolism and in the nucleic acids, antioxidants, lipids and neurotransmitters syntheses (Dhir et al. 2019).There are many clinical manifestations of this vitamin deficiency among of them neurologic, respiratory, metabolic, and cardiovascular disorders which pose challenges for physicians.(Smith et al. 2021).Thiamine and biotin (vitamin B7) transports are performed by two membrane channels THTR1 and THTR2 encoded by SLC19A2 (OMIM 249,270) and SLC19A3 (OMIM 607,483) genes.SLC19A3 variants are responsible for biotin-thiamine responsive basal ganglia disease (BTRBGD), Leigh syndrome (LS), infantile spasms with lactic acidosis and Wernicke-like encephalopathy (Gerards et al. 2013;Lake et al. 2016).In some cases, SLC19A3 variants abrogate thiamine transport, resulting in vitB1 deficiency, and downstream alterations of many cellular pathways.
Here, we report a consanguineous Tunisian family including an affected individual with Leigh syndrome.NGSequencing of a panel of 281 nuclear genes encoding mitochondrial proteins was carried out in the index case and disclosed a homozygous pathogenic SLC19A3 variant (c.1264A > G; p.T422A).To gain insights on the pathogenicity of this variant, we performed the molecular docking of the SLC19A3 amino acid change and assessed the plasma oxidative stress and mtDNA copy number from the patient.

Patient
The present study has been approved by the institutional review board « Hadi Chaker » University hospital ethics committee'' (Sfax, Tunisia).Written informed consent was obtained.
from the parents of the subject, in agreement with the Declaration of Helsinki.
He is a 2-year-old boy from a consanguineous family, born at 39 weeks of gestation following an uncomplicated pregnancy.His psychomotor development was normal.
At the age of 2 years, he developed an acute ataxia with impairment of consciousness.Brain MRI was characterized by bilateral symmetrical increased T2 and decreased T1 signal intensity at the basal ganglia, thalami, brainstem temporal-parieto-occipital cortex, and subcortical white matter and cerebellum (Fig. 1. A, B, C).
Clinical conditions spontaneously improved, but 2 months later he developed acute focal seizures.Both simple laboratory investigations (electrolytes, glucose, calcium, magnesium, phosphorus) and the cerebrospinal fluid analysis (CSF) were normal.His blood lactate level was elevated: 5.2 mmol/L (normal value < 2 mmol/L).Brain MRI examination showed bilateral abnormalities of signal intensity in the lenticular nucleus and in the bilateral periventricular white matter.He also showed abnormal high signal intensity in the bilateral periventricular white matter and the bilateral dentate nucleus (Fig. 1.D, E).The clinical and radiologic pattern was referred as Leigh syndrome or another type of mitochondrial encephalopathy.The boy died one month later at age of 2 and a half years, due to a refractory status epilepticus.

Controls
Fifty healthy individuals from Tunisia with an average age of 2 years, were tested by Q-PCR for mtDNA depletion and deletions and five among them are studied for the oxidative stress in their plasmas.

Library preparation and sequencing
Total DNA was extracted from peripheral blood using phenol chloroform standard procedures (Lewin, 1992).Custom NGS panel of 281 nuclear genes encoding mitochondrial proteins involved in the most frequent mitochondrial pathologies was used to screen for mutations in the index case.Library preparation for each sample was carried out using SureSelect Target Enrichment System for Sequencing on Ion Proton (Manuel Number G7530-90,005) as described as elsewhere (Felhi et al. 2020).
The selected variant was verified by Sanger sequencing.Exon 5 of the SLC19A3 gene was amplified using primer sets F: 5'-CCCTGTGGCCAATATGTTCT − 3'; R: 5'-TTGCTTGTTGTAAGGTTGAGAAA − 3'.PCR was performed with 50 ng of genomic DNA, with an initial denaturation at 94 °C for 3 min, 30 cycles of 94 o C for 30 s, 60 o C for 45 s and 72 o C for 2 min, followed by a final extension at 72 o C for 10 min.The amplified sequence was sequenced directly from purified PCR products using a Big-Dye Terminator Cycle Sequencing kit (version 3.1; Applied Biosystems; Thermo Fisher Scientific, Inc.) before analysis on an ABI 3130 automated DNA sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc.).

MtDNA depletion and deletions
The mtDNA copy number from blood was quantified by real-time quantitative polymerase chain reaction (qPCR) as described elsewhere (Felhi et al. 2020).MtDNA rearrangement and deletions were analyzed using eKLIPse software (Goudenege et al., 2019).

Molecular docking
Modeling of the 3D structure of wild type SLC19A3 protein was done with I-TASSER 5.1 (http://zhanglab.ccmb.med.umich.edu/I-TASSER/).Structure-based function prediction was performed using the COACH method.The two models were optimized using the OPLS-AA force field until the gradient of 0.01 kcal/(Å.mol) was reached (Gschwend et al. 1996).The best-ranked docking pose of each chemical compound in the active site of SLC19A3 was obtained according to the scores and binding-energy value.The similarity between the best docking pose and experimental crystal pose was calculated using the root-mean square deviation (RMSD) (Lee et al., 2020).Receptor-ligand interactions were analyzed and drawn by using the Discovery Studio Visualizer (DSV) developed by Accelrys (BIOvIA, 2016).

Oxydative stress exploration
Protein quantification: Plasma protein contents were determined in alkaline medium to obtain blue color solutions assayed at 490 nm at 37 °C as described by Lowry et al. (1951).Bovine serum albumin (BSA) was used as a standard.
TBARS-MDA assay: As described by Draper and Hadley (1990), malondialdehyde (MDA) is measured with a spectrophotometer at 532 nm by using thiobarbituric acid (TBA), resulting in a TBA-MDA complex (Rajneesh et al. 2008).Using the following equation, results were expressed as a percentage of inhibition for three replicates.

Molecular docking of the mutated protein harboring the A422 amino-acid change
Since SLC19A3 encodes the vitB1 transporter, mutations in this channel are predicted to alter the passage of vitB1 through the cell membrane.Indeed, patients carrying the p.T422A substitution have been reported to have almost no transport activity.
First, we have analyzed the crystallized model of the SLC19A3 protein by I-TASSER.The visualization of the model built by Discovery Studio Visualizer disclosed a clear channel structure with parallel helices.The result showed that p.T422A change is located in a crucial position on an internal helix interacting with vitB1 (Fig. 2

. A1, A2).
To analyze the effect of this variant on the passage of vitB1, we performed and compared the molecular docking between the wild-type and the mutated protein with vitB1.Results showed that the p.T422A leads to conformational changes of the whole protein structure and modifies the interactions with the substrate (Fig. 2. B1, B2).In the wild-type protein, vitB1 forms four hydrogen bonds with T422 amino acid, whereas the mutated A422 amino acid can establish only a single hydrogen bond with vitB1 (Fig. 2. C1, C2).

Oxidative stress markers in patient plasma and healthy controls
VitB1 deficiency alters the reactive oxygen species (ROS) balance at the cellular and mitochondrial levels.We evidenced that the plasma Malondialdehyde (MDA) level is significantly higher (p < 0.01) in the patient compared to control individuals (Fig. 3A), while plasma antioxidant activity associated to CAT, SOD and GSH enzymes were significantly lower (p < 0,05) in the patient compared to controls (Fig. 3.B).

MtDNA analysis
We studied the consequences of the oxidative stress on mtDNA integrity by evaluating possible deletions or depletion.No mtDNA rearrangement or deletion was observed using the eKlipse software (data not showed), but the mtDNA copy number showed a 42% reduction in the patient compared to age-matched controls (Fig. 3.C).

Enzymatic antioxidant activities (superoxide dismutase: SOD; Catalase: CAT):
The SOD and CAT activities were explored in the plasma of the patient and in five healthy agematched individuals.
SOD activity was determined based on the photoreduction of the nitroblue tetrazolium (NBT) using the protocol described by Asada et al. (1974) at 37 °C.Absorbance was recorded at 580 nm.Data were expressed as units/milligram of protein.
Catalase (CAT) activity was analyzed by the decomposition of hydrogen peroxide according to the method described by Aebi (1984)

Statistical analyses
In vitro and in vivo data were expressed as means standard deviation (SD).Statistical analyses were performed using SPSS 23.0 analysis software using the one-way analyses of variance (ANOVA) followed by the Fisher test (Stat View).The significance was accepted at p < 0.05.

Results
In this report, we studied a consanguineous Tunisian family including an affected individual with clinical features suggestive of Leigh syndrome.Next-Generation Sequencing of a panel of 281 nuclear genes encoding mitochondrial proteins was performed in the indexed case (II.1) and variant filtering led to the identification of the homozygous variant c.1264A > G (p. T422A) in exon 5 of SLC19A3.Family segregation by Sanger sequencing disclosed the presence of the variant in the heterozygous state in the parents, and its absence in the healthy brothers (Fig. 1.II).This variant was predicted to be pathogenic according to the ACMG classification (PP3/ PP5/PM2).Furthermore, analyses using I-Mutant and Mutpred softwares suggested a decrease in protein stability and an alteration of the transmembrane protein surface.ganglia disease (BTRBGD, OMIM# 607,483).Previous functional studies confirmed its pathogenicity due to a down-regulation of the THTR2 thiamine transport (Vernau et al., 2015).
Molecular docking studies revealed that the affected amino acid is located on the channel surface and induces a mis-orientation of vitB1 in the mutated protein due to conformational changes induced by the mutation and the decreased affinity of the protein for its ligand leading to an unfavorable environment for vitB1 transport.

Discussion
In the present study, we performed molecular, biochemical and computational studies of samples issued from a Tunisian patient displaying a Leigh syndrome.
NGS sequencing of panel of nuclear genes encoding mitochondrial proteins revealed a known homozygous SLC19A3 variant c.1264A > G (p.T422A), which is predicted to be pathogenic according to the ACMG classification and referenced as responsible for biotin-thiamine responsive basal biosynthesis, including ribose-5P, an essential precursor of nucleotide biosynthesis (Vernau et al., 2015).Thus, the deficiency in NADPH and ribose-5P can explain the increase in oxidative stress and the decrease in mtDNA copy number in the patient.The lower mtDNA copy number observed is supported by previous studies in dogs and seals (Vernau et al., 2015;Croft et al., 2013), leading to an imbalance of nucleotide pools between the cytosol and mitochondria (Fig. 4).Furthermore, various problems in neurotransmission could produce in lack of thiamine, most reported the glutamatergic and GABAergic systems, resulting in a toxic neuroexcitatory state that contributes to the neurologic impairment in all described patients (Butterworth 1989;Todd et al. 1999;Freitas-Silva et al. 2010).
SLC19A3 deficiency was initially reported in patients from Saudi Arabia as biotin-responsive basal ganglia disease (Ozand et al. 1998).These patients presented in childhood with a subacute encephalopathy and symmetrical lesions in the basal ganglia, particularly the caudate nucleus VitB1 is a potent antioxidant, preventing harmful free radical damages and also and also inhibiting lipid peroxidation (Nga and Quang, 2019).Plasma analyses of our patient disclosed a significant increase in MDA, which corresponds to the primary marker of oxidative stress, reflecting abnormal lipid peroxidation.Furthermore, we found a significant decrease in antioxidant enzymes as the SOD and CAT, as well in the nonenzymatic antioxidant GPX.In a previous study carried on dogs, thiamine transport defects caused by SLC19A3 mutations increased oxidative stress (Verneau et al., 2015), which we confirm here in a human case.
Once in the cytosol, thiamine is converted into its active form by thiamine phosphorylase (TPP), generating a cofactor required for several enzymes involved in cell metabolism.Indeed, TPP serves as a cofactor for the pentose phosphate pathway enzyme and transketolase (TK) (Alfadhel et al. 2019).It produces reducing equivalents of NADPH that support fatty acid synthesis, antioxidant defenses, such as the glutathione peroxide-reductase system, and nucleotide and putamen.Today, the spectrum of SLC19A3 variants has expanded to neurologic mitochondrial syndromes, with the most common being LS (Gerards et al. 2013;Ortigoza-Escobar et al. 2014, 2016).The p.T422A variant is a recurrent pathogenic mutation in the Arab region, which was identified with a founder effect in the Saudi Arabian population for BTRBGD (Zeng et al. 2005;Alfadhel et al. 2013Alfadhel et al. , 2019) ) and was recently reported in a Tunisian LS patient (Hechmi et al. 2022).Supplementation of thiamine and biotin can be effective in treating patients harboring deficiency in thiamine transporter genes such as SLC19A3 and SLC25A19, since it can increase intracellular thiamine levels.Thus, this disease is potentially treatable, but only if the genetic diagnosis is provided early at the onset of the disease.Indeed, among patients who received vitB1 treatment within 1 month after the disease onset, 73.3% of them displayed a good recovery, becoming either symptom-free or with mild deficits, while this proportion decreased to 35.0% with patients that received a delayed therapy (Wang et al., 2021).Individualized doses according to the variant impact on vitB1 deficiency should be recommended for patients.Indeed, patients with mutations that impair vitB1 passage should be treated differently as those with mutations that completely block the passage.Unfortunately, our patient did not benefit from thiamine and biotin supplementation, because the genetic diagnosis was obtained too late.He was admitted to the hospital pediatric service at the age of 18 months for ADEM (Acute Disseminated Encephalomyelitis and Encephalitis).The brain MRI performed at the age of 2 years showed basal ganglia and white matter involvement suggesting a Leigh syndrome diagnosis and he passed away after few months.

Conclusion
In summary, we reported a patient from a consanguineous Tunisian family with a clinical presentation of LS, who harbored the founding Arab pathogenic SLC19A3variant p.T422A affecting vitB1 transport.Further modelization of the mutation impacts and biochemical analyses confirmed the pathogenicity of the variant and its involvement in LS.

Fig. 1
Fig. 1 (I): Brain MRI of the patient at the age of 2 years: Bilateral and symmetrical decreased signal intensity in T1-weighted images (A) with increased signal intensity in axial T2-weighted images (B, C) in basal ganglia, thalamus, splenium of corpus callosum, midbrain, temporal-parieto-occipital cortex, and subcortical white matter and cerebellum.Diffusion weighted imaging: b1000 images (D, E) show-

Fig. 2
Fig. 2 Molecular modeling of SLC19A3 transporter and the docking of wild type (T422) and mutated model (A422).(A1) modeling structure of the SLC19A3 channel into the phospholipidic membrane.(A2): location of the affected amino acid within the channel, (B1).(C) molecular docking showing the distribution and location of the amino acids interacting with vitB1 in the wild type (B1) and the mutated model (B2), (C) binding of the wild type A422 (C1) and mutated T422 (C2) amino acid to vitB1

Fig. 3
Fig. 3 (A): Dosage of lipid peroxidation (MDA), (B) Dosage of antioxidant activities: SOD, CAT and GSH in patient's plasma compared to control plasmas (C) Quantification of mtDNA copy number in patient and controls

Fig. 4
Fig. 4 Impact of SLC19A3 mutation on the metabolism related to VitB1/thiamine: Impact on mtDNA content and oxydative stress state at 37 °C.A decrease in absorbance due to H 2 O 2 degradation was monitored at 240 nm for 1 min.Results were expressed as micromoles of H 2 O 2 consumed/milligram of protein.