Report of a novel recurrent homozygous variant c.620A>T in three unrelated families with thiamine metabolism dysfunction syndrome 5 and review of literature

Abstract

Introduction:

Biallelic variants in thiamine pyrophosphokinase 1 (TPK1) are known to cause thiamine metabolism dysfunction syndrome 5 (THMD5). This disorder is characterized by neuroregression, ataxia, and dystonia with basal ganglia abnormalities on neuroimaging. To date, 27 families have been reported with THMD5 due to variants in TPK1.

Methods:

We ascertained three individuals from three unrelated families. Singleton exome sequencing was performed on all three individuals, followed by in silico mutagenesis of the mutant TPK protein. Additionally, we reviewed the genotypic and phenotypic information of 27 previously reported individuals with THMD5.

Results:

Singleton exome sequencing revealed a novel homozygous variant c.620A>T p.(Asp207Val) in TPK1 (NM_022445.4) in all three individuals. In silico mutagenesis of the mutant protein revealed a decrease in protein stability and altered interactions with its neighboring residues compared to the wild-type protein. Thus, based on strikingly similar clinical and radiological findings compared to the previously reported individuals and with the support of in silico mutagenesis findings, the above-mentioned variant appears to be the probable cause for the condition observed in the affected individuals in this study.

Conclusion:

We report a novel homozygous variant in TPK1, which appears to be recurrent among the Indian population.

Introduction

Thiamine or vitamin B1 (also known as aneurine), is an essential water-soluble nutrient that plays an important role in various cellular pathways of energy metabolism. In humans, thiamine is available as free-thiamine, thiamine pyrophosphate (TPP), thiamine monophosphate (TMP) and thiamine triphosphate (TTP) (Marcé-Grau et al., 2019). Free-thiamine and TMP account for a very low percentage of total thiamine in humans (Losa et al., 2005). Free-thiamine is prominently found in central nervous system (CNS) and is involved in generation of acetylcholine (Manzetti, Zhang and van der Spoel, 2014), uptake of serotonin (Vigil et al., 2010) and gamma-aminobutyric acid (Ferreira-Vieira et al., 2016), and as an antioxidant for reactive species of nitrogen and oxygen (Huang et al., 2010). It is also involved in the regulation and activation of brain immune cells and the expression of antibodies, immunoglobulins, and CD40L-mediated immune system (Manzetti et al., 2014). TTP accounts for 5–10% of total body thiamine and acts as a phosphate donor in the phosphorylation reaction of energy metabolism (Losa et al., 2005; Mkrtchyan et al., 2015). TPP accounts for 80% of total body thiamine (Losa et al., 2005) and takes part in multiple metabolic processes across the cytosol, mitochondria, and peroxisome.

The uptake of thiamine across the plasma membrane takes place by active transport via thiamine transporter 1 encoded by SLC19A2 and thiamine transporter 2 encoded by SLC19A3 (Marcé-Grau et al., 2019). It is later phosphorylated by the enzyme thiamine pyrophosphokinase (TPK) encoded by TPK1, to produce its active form TPP (Ortigoza-Escobar et al., 2017). TPP is a cofactor for thiamine-dependent transketolase in the pentose phosphate pathway in cytosol. In mitochondria, TPP is transported by a mitochondrial carrier encoded by SLC25A19, where it acts as a cofactor for several mitochondrial enzyme complexes (Mayr et al., 2011). In the peroxisomes, TPP acts as a cofactor for 2-hydroxyacyl-CoA lyase involved in fatty acids degradation (Ortigoza-Escobar et al., 2016) (Figure S1).

Thiamine deficiency manifest due to acquired deficiency (Beriberi and Wernicke’s encephalopathy) predominantly affecting CNS and cardiovascular system in vulnerable population (Marcé-Grau et al., 2019) or due to defects in gene encoding proteins with functions in thiamine transport and metabolism leading to thiamine metabolism dysfunction syndrome (THMD). To date, variants in four genes are known to be associated with THMD. SLC19A2 variants cause thiamine-responsive megaloblastic anemia syndrome (also known as THMD1, MIM #607483), an early onset disorder characterized by sensorineural deafness, diabetes mellitus and megaloblastic anemia (Labay et al., 1999). Variants in SLC19A3 cause THMD2 (MIM #607483), with clinical manifestations of sub-acute encephalopathy, seizures, external ophthalmoplegia, dysphagia and dystonia (Ozand et al., 1998; Zeng et al., 2005). SLC25A19 is associated with Amish-type microcephaly (also known as THMD3, MIM #607196) and THMD4 (MIM #13710). Amish-type microcephaly is characterized by severe microcephaly, episodic encephalopathy associated with lactic acidosis, profound delayed psychomotor development and alpha-ketoglutaric aciduria whereas THMD4 characterized by childhood-onset recurrent episodes of encephalopathy associated with febrile illnesses and slow chronically progressive axonal polyneuropathy (Kelley et al., 2002; Spiegel et al., 2009). Deficiency of TPK due to biallelic variants in TPK1 causes THMD5 (MIM #614458), where individuals clinically manifest recurrent episodic encephalopathy, psychomotor retardation, variable degrees of ataxia, progressive dystonia with basal ganglia and cerebellar abnormalities on neuroimaging (Mayr et al., 2011).

We herein report three individuals from three unrelated families with a novel recurrent homozygous variant in TPK1 and highlight the clinical phenotype with treatment strategies available for early detection and management of disorder with thiamine metabolic dysfunction. We also provide a phenotypic and genotypic review of the previously reported individuals.

Methods

Clinical report

Proband 1 (P1) is a two-year-old male born from a consanguineous union of family 1 (Figure 1A). His development was age appropriate until one year of age, after which he had neuroregression. He could walk for short distances, associated with frequent falls. At two years of age, he developed sudden onset fever followed by stereotypic movements of the mouth throughout the day and his vocabulary was restricted to monosyllables with low social skills. His elder sibling was documented to have similar clinical presentation as him at approximately same age and succumbed to death at three years of age. Upon examination at two-years, his weight was 9.7kg (−4.67SD), height was 90cm (−4.40SD) and head circumference was 48cm (−2.33SD). He had dystonic movements in upper limbs. The sensory and motor system were intact on examination, and fundus was normal with no organomegaly. On magnetic resonance imaging (MRI) of brain, T2-weighted hyperintensities in the putamen and caudate were observed (Figure 1Di–ii). Dextrose, lactate, and arterial blood gas (ABG) investigations were normal with unremarkable tandem mass spectrometry (TMS) and gas chromatography mass spectrometry (GCMS) for screening of metabolic disorders.

Figure 1.

(A-C) Pedigrees for family 1, 2 and 3, respectively (D) Magnetic resonance imaging (MRI) showing (Di - vi) T2-weighted/FLAIR hyperintensities in caudate and putamen in proband 1, 2 and 3 (P1, P2 and P3), respectively. In addition, (Diii-iv) P2 had hyperintensities in the periventricular white matter and (Dv-vi) P3 showed mild cerebral volume loss and cortical atrophy. (E) Multiple sequence alignment (MSA) of TPK sequences showing the 207^th position is not conserved across species. The asterisk (*) denotes a fully conserved residue, colon (:) denotes residues with strongly similar properties and period (.) denotes residues with weakly similar properties. (F) Structure of human TPK showing two chains, A (red) and B (dark blue) with thiamine pyrophosphate (TPP) (green) bound to it. (Fi) In the wild-type protein, Asp207 (yellow) has polar contacts (orange lines) with Val195 (pink) and Thr205 (light blue). (Fii) In silico mutagenesis revealed loss of polar contact with Thr205 in the mutant TPK protein. (G) Schematic representation of TPK1 gene with all reported variants along with the variant reported in our study represented in red.

Proband 2 (P2) is 21-month-old male born to non-consanguineous parents from family 2 (Figure 1B). At six months of age, he was noted to have mild developmental delay with head control achieved at six months, sitting with support at seven months, sitting without support at nine months, standing with support at one year and speaking bisyllables at one year of age. At 13 months of age, he had febrile convulsions which was focal in nature involving left upper limb followed by a second episode of seizure which involved left upper and lower limbs along with dystonic posturing of right limb. The seizures were followed by regression of developed milestones. On examination at one year eight months, his weight was 8.6 kg (−1.69SD), length was 86 cm (+3.36SD), and head circumference was 47 cm (+0.50SD). He had bilateral upper limb intermittent dystonia and lower limb hypotonia. Drooling of saliva and dysphagia were present. He was able to follow light and sound. Electroencephalogram (EEG), liver function test, complete blood count, TMS and GCMS showed normal results. MRI of brain showed T2-weighted/FLAIR hyperintensities in putamen, caudate and posterior periventricular white matter (Figure 1Diii–iv).

Proband 3 (P3) is a three-year-old female born from a non-consanguineous union of family 3 (Figure 1C). She attained motor milestones appropriate for age, however a significant language delay was noted. At two years of age, she was hospitalized for bronchopneumonia after which regression of milestones was noted. The second episode of seizure was documented at three years of age. On examination at three years of age, her weight was 8.14 kg (−4.79SD) and head circumference was 45 cm (−2.66SD). She had opisthotonic posturing, dystonia, and spasticity in all four limbs with hypersensitivity to physical stimulus. Her reflexes were exaggerated, and extensor plantar was observed. No obvious facial dysmorphism and organomegaly was seen. She followed commands and eye contact was positive. Blood creatine phosphokinase and lactate were 119 U/L (34–149 U/L) and 12.3 mg/dL (5–20 mg/dL), respectively. EEG and ophthalmologic evaluation showed normal results. MRI brain showed bilateral hyperintensities in caudate and putamen along with mild cerebral volume loss and cortical atrophy (Figure 1Dv–vi). She was treated with biotin (80mg /day) and thiamine (500mg/day) supplements daily, however she succumbed to death at four years of age. She had a similarly affected elder sibling, with seizures followed by neuroregression and MRI was suggestive of hypoxic ischemic encephalopathy. However, samples were unavailable for genetic testing as he succumbed to death at two years seven months of age due to pneumonia.

Molecular testing

Informed consents approved by the institutional ethics committee, Kasturba Medical College and Kasturba Hospital were obtained from the recruited families. Genomic DNA was isolated from whole blood of the families using QIAamp DNA kit (QIAGEN, Valencia, California, USA). Singleton exome sequencing (ES) (Illumina, Inc. San Diego, California) was performed for all the probands (P1, P2 and P3) from three unrelated families. Quality assessment and processing of the data, variant calling and annotation followed by analysis was done as described previously (Kausthubham et al., 2021). Details of the capture kits used are given in the supplementary material. Sanger sequencing was done for segregation analysis and validation of the disease-causing variant in the families.

In silico protein modelling and analysis

Multiple sequence alignment (MSA) was done using ClustalOmega. The TPK protein structure, obtained from Protein data Bank (PDB) (PDB ID: 3S4Y; accessed on March 24th, 2023) was loaded into UCSF Chimera (v1.16) (Pettersen et al., 2004), which was followed by in silico mutagenesis of the missense variant p.(Asp207Val) to obtain the mutant protein. Polar contacts were determined between the wild-type and its neighboring amino acids, and we compared it with the mutant protein. The structural consequences of the mutant amino acid Asp207Val was also predicted using HOPE and I-Mutant.

Results

Singleton ES revealed a novel homozygous missense variant c.620A>T p.(Asp207Val) in TPK1 (NM_022445.4) in all three probands. Sanger sequencing confirmed the variant in homozygous state in the probands and heterozygous state in their parents. In silico tools such as MutationTaster, SIFT, Polyphen2 and FATHMM_MKL predicted the variant as benign and, REVEL, CADD Phred and GERP++ scores were 0.144, 17.16 and −0.692, respectively. The variant was absent in homozygous state in population databases ExAC (Exome Aggregation Consortium), gnomAD and our in-house database of 2576 exomes. It was classified as variant of uncertain significance as per the American College of Medical Genetics and Genomics guidelines (Richards et al., 2015).

We carried out MSA, which showed that the 207^th position was not conserved across species (Figure 1E). In silico mutagenesis and analysis of the TPK protein was done to determine the effects of the missense variant Asp207Val. In the wild-type protein, Asp207 has polar contacts with Thr205 and Val195. Due to the missense variant, there is loss of interaction with Thr205 in the mutant protein (Figure 1Fi–ii).

According to the predictions done by HOPE, based on size and charge, the mutant residue is smaller and neutral in charge compared to the wild type, which is larger and negatively charged. Also, based on the location of the mutated residue, it is present in the domain that is predicted to be involved in thiamine binding. I-Mutant predicted Asp207Val to cause decrease in protein stability (−0.45 Kcal/mol DDG) in the mutant protein.

Discussion

Thiamine metabolism dysfunction syndrome 5 (THMD5) is a rare yet treatable neurodegenerative disorder caused due to biallelic variants in TPK1 gene. To date, there are 27 individuals from 21 unrelated families reported with biallelic variants in TPK1 (Table 1, Supplementary table 1). In all affected individuals, symptoms manifested during infancy or early childhood. Most individuals initially demonstrated normal neurological development, but later experienced deterioration characterized by neuroregression, ataxia, seizures, dystonia, and spasticity which were often triggered by febrile illnesses (Rüsch et al., 2021). However, few individuals showed delayed development of motor and cognitive functions before the acute manifestations of TPK deficiency (Mayr et al., 2011; Banka et al., 2014; Fraser et al., 2014; Bugiardini et al., 2019; D. Li et al., 2020; X. Li et al., 2022; Eckenweiler et al., 2021; van der Ven et al., 2021). Neuroimaging findings were predominantly marked by hyperintensities in the basal ganglia, dentate nuclei, and thalamus, also cerebral atrophy was present in few individuals (Marcé-Grau et al., 2019). In congruence to the clinical manifestations seen in previously reported individuals, the three individuals in our study had neuroregression, spasticity and dystonia triggered by viral infection or febrile seizures. MRI findings were also consistent with previously reported individuals.

Table 1:

Overview of phenotype and genotype of individuals with THMD5

	This study			Previous studies
	P1	P2	P3
Number of individuals (families)	3 (3)			27 (21)
Variant in TPK1 (NM_022445.4)	c.620A>T p.(Asp207Val)			Missense = 18 (25) Frameshift = 2 (25) Splicing = 4 (25) Gross deletion = 1 (25)
Zygosity	Homozygous			Homozygous = 13 (27) Compound heterozygous = 14 (27)
Gender	Male	Male	Female	Female = 13 (25), Male = 12 (25)
Age last examined	2 years 6 months	2 years	3 years	11 months to 40 years
Age at onset	1 year	13 months	1 year	2 days to 7 years
Triggers*	+	+	−	14 (24)
Clinical examination
Motor delay	−	+	−	8 (19)
Cognitive delay	−	−	−	5 (20)
Regression (Age)	+ (1 year)	+ (13 months)	+ (1 year)	14 (19)
Speech delay/disorder	+	+	+	11 (17)
Seizures	−	+	+	6 (17)
Feeding difficulty	−	+	−	4 (16)
Encephalopathy	−	−	−	11 (22)
Hypotonia	−	+	−	8 (19)
Hypertonia	−	−	+	13 (19)
Dystonia	+	+	+	9 (21)
Gait abnormalities	+	−	−	22 (18)
Involuntary movements	+	−	−	3 (16)
Magnetic resonance imaging (MRI)
Cerebral atrophy	−	−	+	5 (24)
Hyperintensities in basal ganglia	+	+	+	21 (24)
Cerebellum**	−	−	−	11 (24)
Brainstem (Medulla and Pons)	−	−	−	3 (24)

⁺, present;

⁻, absent;

^*,triggers for onset of clinical manifestations in the form of viral infections, fever or febrile seizure;

^**, hyperintensities in dentate nuclei

With a considerable overlap in clinical features of TPK deficiency and other inborn errors of metabolism, establishing a diagnosis based on clinical symptoms alone can be very challenging. Thereby, making biochemical testing with genetic analysis crucial for accurate identification of the disease. Many previously reported individuals with TPK deficiency commonly exhibited elevated levels of lactate in both serum and cerebrospinal fluid (CSF), along with variable elevation of α-ketoglutarate levels in urine (Supplementary table 1). In addition, plasma thiamine levels were either normal or reduced but the blood TPP levels were significantly reduced. Therefore, measurement of blood TPP and urinary alpha-ketoglutaric acid levels will further aid in establishing the diagnosis of THMD5. Unfortunately, this method is not widely accessible in most clinical laboratories.

TPK1 gene is located at 7q35 and contains nine exons, which encodes for TPK protein of 243 amino acids (B. Zhu et al., 2020). TPK is a homodimer which contains a dimerization domain and thiamine binding domain. The dimer interface of two TPK molecules forms the binding site for thiamine (Banka et al., 2014; Marcé-Grau et al., 2019). There are 25 variants reported in TPK1, which includes missense, frameshift, splicing variants, and a copy number variant (exon 3 and 4 deletion) (Table 1, Supplementary table 1) (Figure 1G). On ES analysis, a missense variant c.620A>T p.(Asp207Val) in TPK1 in homozygous state was found in three individuals in our study. In silico tools predicted the variant as benign and MSA inferred the 207^th position was not conserved across species. However, given the remarkably similar clinical and radiological findings compared to the previously reported individuals, backed by in silico mutagenesis results, the variant c.620A>T appeared to be the most probable cause for the condition observed in the affected individuals in our study.

Various functional and biochemical studies were done to establish the pathogenicity of previously reported TPK1 variants and to provide an insight of the mechanisms causing the disease (Table 2). In silico predictions of Asp207Val was suggestive of it being present in the thiamine binding domain, and we hypothesize that this could affect the efficiency of thiamine binding and thereby impair the downstream processes, however further functional validation is required to validate this hypothesis.

Table 2:

Various functional and biochemical assays carried out for previously reported TPK1 variants.

Reference	Functional/Biochemical assay	Variant in TPK1	Results
(Mayr et al., 2011)	Metabolic flux and mitochondrial enzyme assay from muscle biopsy	c.[148A>C];[501+4A>T] p.[(Asn50His)];[(Val119_Pro167)]	Defects in the mitochondrial pyruvate oxidation and metabolism of thiamine
		c.119T>C p.(Leu40Pro)
		c.[179_182delGAGA];[656A>G] p.[(Arg60Lysfs*52)];[(Asn219Ser)]
	Concentration of thiamine, TMP and TPP in fibroblast, muscle and blood sample, post treatment with thiamine supplements	c.[148A>C];[501+4A>T] p.[(Asn50His)];[(Val119_Pro167)]	Reduced TPP levels in muscle and blood
		c.119T>C p.(Leu40Pro)	Reduced thiamine, TMP and TPP levels in muscle and blood. Reduced TPP levels in fibroblasts
		c.[179_182delGAGA];[656A>G] p.[(Arg60Lysfs*52)];[(Asn219Ser)]	Reduced thiamine, TMP and TPP levels in muscle and blood
(Banka et al., 2014)	Mitochondrial enzyme assay from muscle biopsy	c.664G>C p.(Asp222His)	Reduced utilization of pyruvate but normal PDHC activity
	Quantification of TPP from muscle and blood	c.479C>T p.(Ser160Leu)	Reduced TPP levels
		c.664G>C p.(Asp222His)
	Transfection of E.coli cells with wild-type and mutant TPK1 followed by TPK enzyme activity assay	c.479C>T p.(Ser160Leu)	Reduced TPK enzyme activity
		c.664G>C p.(Asp222His)
	Quantification of TPK from muscle extracts	c.479C>T p.(Ser160Leu)	Normal
		c.664G>C p.(Asp222His)	Decrease in TPK levels
(Fraser et al., 2014)	Plasma amino acid and electron transfer studies from muscle biopsy	c.604T>G p.(Trp202Gly)	Inconclusive
	Pyruvate carboxylase and pyruvate dehydrogenase level assay from fibroblast
(Huang et al., 2019)	In vitro thiamine binding, TPK enzyme activity and TPK protein stability assays	c.83A>G p.(Leu28Ser)	Normal thiamine binding, TPK levels and enzymatic activity compared to controls
	In vitro thiamine binding assay of previously reported variants	c.479C>T p.(Ser160Leu) and c.604T>G p.(Trp202Gly)	Partial and reduced thiamine binding, respectively
	In vitro TPK enzyme activity assay of previously reported variants	c.(148A>C) p.(Asn50His)	Reduced enzyme activity
		c.479C>T p.(Ser160Leu)  and c.604T>G p.(Trp202Gly)	Increased enzyme activity
	In vitro TPK protein stability assay of previously reported variants	c.479C>T p.(Ser160Leu), c.604T>G p.(Trp202Gly), c.656A>G p.(Asn219Ser) and c.664G>C p.(Asp222His)	Reduced protein stability
(Bugiardini et al., 2019)	Quantification of TPK protein level from fibroblasts	c.[426G>C];[258+1G>A] p.[(p.Leu142Phe)];[(?)]	Reduced TPK protein levels
(Eckenweiler et al., 2021)	TPK protein quantification from fibroblasts, post treatment with biotin and thiamine	c.[501+4A>T];[479C>T] p.[(Val119_Pro167)];[(Ser160Leu)]	Reduced TPK protein levels

TPP, thiamine pyrophosphate; TMP, thiamine monophosphate; TPK, thiamine pyrophosphokinase; PDHC, pyruvate dehydrogenase complex

THMD5 is treatable with early diagnosis and management. Supplementation of thiamine (100–500mg/day), has led to clinical improvement and near normal neurodevelopment in patients treated at early age (Marcé-Grau et al., 2019) (Supplementary table 1) and has proven to prevent further metabolic decompensations and improve brain MRI lesions (Banka et al., 2014; Invernizzi et al., 2017; X. Li et al., 2022). However, some individuals did not show improvement post thiamine supplementation which could be due to supplements provided at later time, low dosage of thiamine applied or severe clinical presentation before the initiation of treatment (Mahajan and Sidiropoulos, 2017; Bugiardini et al., 2019; L. Zhu et al., 2019; Au et al., 2020). Also, supplementation with higher doses of thiamine at early age of detection proved to have a significant improvement in clinical condition and also prevention of encephalopathic episodes (Banka et al., 2014; Fraser et al., 2014).

In conclusion, we report a novel homozygous variant in TPK1 in three unrelated families, which appears to be recurrent and common among the Indian population. We suggest screening for this variant in patients with recurrent episodes of encephalopathy, ataxia and/or dystonia, MRI brain suggestive of hyperintensities in basal ganglia along with high serum and CSF lactate or increased α-ketoglutarate in urine. In addition, early intervention has led to potential benefits in patients with THMD5. High suspicion, definitive diagnosis and early management with essential vitamins will help in prevention and progression of the debilitating adversities associated with TPK deficiency.

Data availability statement

The data providing the evidence of the study is available from the corresponding author upon reasonable request.