Thiamine metabolism dysfunction syndrome 5 (THMD5) mimicking acute disseminated encephalomyelitis.

Thiamine pyrophosphate (TPP), the substrate of Thiamine pyrophosphate kinase (TPK), is an important cofactor in carbohydrate metabolism, specifically as a cofactor of the Pyruvate dehydrogenase complex (PDH) complex. The nervous system is particularly dependent on TPP due to its reliance on glucose metabolism. In this case, a four-year-old girl had a previously unreported pathogenic variant of the gene encoding TPK (TPK1) which presented as Thiamine metabolism dysfunction syndrome 5 (THMD5; OMIM 614458). She had been diagnosed with acute disseminated encephalomyelitis and autism spectrum disorder (ASD), and initially presented with fever and agitation following vaccinations. After follow-up with genetic testing, our patient was found to have compound heterozygous pathogenic variants of TPK1. After treatment with biotin and thiamine her clinical status improved, and her ASD features resolved. The presentation of our patient was consistent with previous reports and adds to the evidence that thiamine and biotin are effective treatments of TPK1 related metabolic deficiencies. The improvement of neurobehavioral symptoms in this case was marked, highlighting the importance of early identification and therapeutic intervention in this condition.

Case report of two affected siblings in a family with thiamine metabolism dysfunction syndrome 5: a rare, but treatable neurodegenerative disease.

BACKGROUND: Thiamine metabolism dysfunction syndrome 5 (THMD5) is a rare inherited metabolic disorder due to thiamine pyrophosphokinase 1(TPK1) deficiency, caused by mutations in TPK1. The core symptoms of the disease is acute or subacute onset encephalopathy, ataxia, muscle hypotonia, and regression of developmental milestones in early infancy, repeatedly triggered by acute infectious illness. However, we report two brothers of THMD5 with compound heterozygous for the mutations c.614-1G > A,c.224 T > A p.(Ile75Asn), but the prognosis is quite different if thiamine suppled. According to our current knowledge, the missense variant c.224 T > A p.(Ile75Asn) was not published previously. CASE PRESENTATION: Here, we describe two affected siblings in a Chinese family, after an uneventful pregnancy to non-consanguineous and healthy parents. The older brother presented with normal development during the first 6 months of life, but developed regression of developmental milestones after, accompanied with muscle hypotonia, and chronic encephalopathy, and died at 1 year and 6 months old. The younger brother presented with acute onset encephalopathy, ataxia, muscle hypotonia, repeatedly triggered by acute infectious illness. He was compound heterozygous for the mutations c.614-1G > A,c.224 T > A p.(Ile75Asn) identified by whole exome sequencing. He was diagnosed of THMD5 when he was 11 month. Oral supplementation of thiamine 100 mg/day, the symptoms gradually disappeared. At the age of 2 years and 4 months, he stoped thiamine, his symptoms returned and were once again relieved by oral supplementation of thiamine 100 mg/day. CONCLUSIONS: THMD5 is a rare, but treatable neurodegenerative disease, the clinical phenotype ranges from mild to severe. Massive-dose of thiamine supplementation may ameliorate the course of TPK1 deficiency. When similar clinical cases appear, gene detection is particularly important, which is conducive to early diagnosis. Treatment with thiamine while awaiting the outcome of diagnostic tests may be a good choice.

Deletion of yeast TPK1 reduces the efficiency of non-homologous end joining DNA repair.

Non-homologous end joining (NHEJ) is a highly conserved mechanism of DNA double-stranded break (DSB) repair. Here we utilize a computational protein-protein interaction method to identify human PRKACB as a potential candidate interacting with NHEJ proteins. We show that the deletion of its yeast homolog, TPK1 that codes for the protein kinase A catalytic subunit reduces the efficiency of NHEJ repair of breaks with overhangs and blunt ends in plasmid-based repair assays. Additionally, tpk1Δ mutants showed defects in the repair of chromosomal breaks induced by HO-site specific endonuclease. Our double deletion mutant analyses suggest that TPK1 and YKU80, a key player in NHEJ could function in parallel pathways. Altogether, here we report a novel involvement for TPK1 in NHEJ.

Reduced thiamine binding is a novel mechanism for TPK deficiency disorder.

Thiamine pyrophosphokinase (TPK) converts thiamine (vitamin B1) into thiamine pyrophosphate (TPP), an essential cofactor for many important enzymes. TPK1 mutations lead to a rare disorder: episodic encephalopathy type thiamine metabolism dysfunction. Yet, the molecular mechanism of the disease is not entirely clear. Here we report an individual case of episodic encephalopathy, with familial history carrying a novel homozygous TPK1 mutation (p.L28S). The L28S mutation leads to reduced enzymatic activity, both in vitro and in vivo, without impairing thiamine binding and protein stability. Thiamine supplementation averted encephalopathic episodes and restored the patient's developmental progression. Biochemical characterization of reported TPK1 missense mutations suggested reduced thiamine binding as a new disease mechanism. Importantly, many disease mutants are directly or indirectly involved in thiamine binding. Thus, our study provided a novel rationale for thiamine supplementation, so far the major therapeutic intervention in TPK deficiency.

Genetic defects of thiamine transport and metabolism: A review of clinical phenotypes, genetics, and functional studies.

Thiamine is a crucial cofactor involved in the maintenance of carbohydrate metabolism and participates in multiple cellular metabolic processes within the cytosol, mitochondria, and peroxisomes. Currently, four genetic defects have been described causing impairment of thiamine transport and metabolism: SLC19A2 dysfunction leads to diabetes mellitus, megaloblastic anemia and sensory-neural hearing loss, whereas SLC19A3, SLC25A19, and TPK1-related disorders result in recurrent encephalopathy, basal ganglia necrosis, generalized dystonia, severe disability, and early death. In order to achieve early diagnosis and treatment, biomarkers play an important role. SLC19A3 patients present a profound decrease of free-thiamine in cerebrospinal fluid (CSF) and fibroblasts. TPK1 patients show decreased concentrations of thiamine pyrophosphate in blood and muscle. Thiamine supplementation has been shown to improve diabetes and anemia control in Rogers' syndrome patients due to SLC19A2 deficiency. In a significant number of patients with SLC19A3, thiamine improves clinical outcome and survival, and prevents further metabolic crisis. In SLC25A19 and TPK1 defects, thiamine has also led to clinical stabilization in single cases. Moreover, thiamine supplementation leads to normal concentrations of free-thiamine in the CSF of SLC19A3 patients. Herein, we present a literature review of the current knowledge of the disease including related clinical phenotypes, treatment approaches, update of pathogenic variants, as well as in vitro and in vivo functional models that provide pathogenic evidence and propose mechanisms for thiamine deficiency in humans.

Expanding the clinical and molecular spectrum of thiamine pyrophosphokinase deficiency: a treatable neurological disorder caused by TPK1 mutations.

Thiamine pyrophosphokinase (TPK) produces thiamine pyrophosphate, a cofactor for a number of enzymes, including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. Episodic encephalopathy type thiamine metabolism dysfunction (OMIM 614458) due to TPK1 mutations is a recently described rare disorder. The mechanism of the disease, its phenotype and treatment are not entirely clear. We present two patients with novel homozygous TPK1 mutations (Patient 1 with p.Ser160Leu and Patient 2 with p.Asp222His). Unlike the previously described phenotype, Patient 2 presented with a Leigh syndrome like non-episodic early-onset global developmental delay, thus extending the phenotypic spectrum of the disorder. We, therefore, propose that TPK deficiency may be a better name for the condition. The two cases help to further refine the neuroradiological features of TPK deficiency and show that MRI changes can be either fleeting or progressive and can affect either white or gray matter. We also show that in some cases lactic acidosis can be absent and 2-ketoglutaric aciduria may be the only biochemical marker. Furthermore, we have established the assays for TPK enzyme activity measurement and thiamine pyrophosphate quantification in frozen muscle and blood. These tests will help to diagnose or confirm the diagnosis of TPK deficiency in a clinical setting. Early thiamine supplementation prevented encephalopathic episodes and improved developmental progression of Patient 1, emphasizing the importance of early diagnosis and treatment of TPK deficiency. We present evidence suggesting that thiamine supplementation may rescue TPK enzyme activity. Lastly, in silico protein structural analysis shows that the p.Ser160Leu mutation is predicted to interfere with TPK dimerization, which may be a novel mechanism for the disease.

Identification of novel mutations in TPK1 and SLC19A3 genes in families exhibiting thiamine metabolism dysfunction syndrome.

Background and aims: The occurrence of thiamine metabolism dysfunction syndrome (THMD), a rare autosomal recessive condition, may be linked to various mutations found in the TPK1 and SLC19A3 genes. The disease chiefly manifests through ataxia, muscle hypotonia, abrupt or subacute onset encephalopathy, and a decline in developmental milestones achieved during the early stages of infancy. We present findings from an investigation that involved two individuals from Iran, both of whom experienced seizures along with ataxia and hypotonia. The underlying genetic causes were found with the use of next-generation sequencing (NGS) technology, which has facilitated the detection of causal changes in a variety of genetic disorders. Material and methods: The selection of cases for this study was based on the phenotypic and genetic information that was obtainable from the Center for Comprehensive Genetic Services. The genetic basis for the problems observed among the participants was determined through the application of whole-exome sequencing (WES). Subsequently, sanger sequencing was employed as a means of validating any identified variations suspected to be causative. Results: The first patient exhibited a homozygous mutation in the TPK1 gene, NM_022445.4:c.224 T > A:p.I75 N, resulting in the substitution of isoleucine for asparagine at position 75 (p.I75 N). In our investigation, patient 2 exhibited a homozygous variant, NM_025243.4:c.1385dupA:pY462X, within the SLC19A3 gene. Conclusions: Collectively, when presented with patients showcasing ataxia, encephalopathy, and basal ganglia necrosis, it is essential to account for thiamine deficiency in light of the potential advantages of prompt intervention. At times, it may be feasible to rectify this deficiency through the timely administration of thiamine dosages. Accordingly, based on the results of the current investigation, these variations may be useful for the diagnosis and management of patients with THMD.

Tomato SlWRKY3 Negatively Regulates Botrytis cinerea Resistance via TPK1b.

Botrytis cinerea is considered the second most important fungal plant pathogen, and can cause serious disease, especially on tomato. The TPK1b gene encodes a receptor-like kinase that can positively regulate plant resistance to B. cinerea. Here, we identified a tomato WRKY transcription factor SlWRKY3 that binds to the W-box on the TPK1b promoter. It can negatively regulate TPK1b transcription, then regulate downstream signaling pathways, and ultimately negatively regulate tomato resistance to B. cinerea. SlWRKY3 interference can enhance resistance to B. cinerea, and SlWRKY3 overexpression leads to susceptibility to B. cinerea. Additionally, we found that B. cinerea can significantly, and rapidly, induce the upregulation of SlWRKY3 expression. In SlWRKY3 transgenic plants, the TPK1b expression level was negatively correlated with SlWRKY3 expression. Compared with the control, the expression of the SA pathway marker gene PR1 was downregulated in W3-OE plants and upregulated in W3-Ri plants when inoculated with B. cinerea for 48 h. Moreover, SlWRKY3 positively regulated ROS production. Overall, SlWRKY3 can inhibit TPK1b transcription in tomato, and negatively regulate resistance to B. cinerea by modulating the downstream SA and ROS pathways.

Protein Kinase A Negatively Regulates the Acetic Acid Stress Response in S. cerevisiae.

Bioethanol fermentation from lignocellulosic hydrolysates is negatively affected by the presence of acetic acid. The budding yeast S. cerevisiae adapts to acetic acid stress partly by activating the transcription factor, Haa1. Haa1 induces the expression of many genes, which are responsible for increased fitness in the presence of acetic acid. Here, we show that protein kinase A (PKA) is a negative regulator of Haa1-dependent gene expression under both basal and acetic acid stress conditions. Deletions of RAS2, encoding a positive regulator of PKA, and PDE2, encoding a negative regulator of PKA, lead to an increased and decreased expression of Haa1-regulated genes, respectively. Importantly, the deletion of HAA1 largely reverses the effects of ras2∆. Additionally, the expression of a dominant, hyperactive RAS2A18V19 mutant allele also reduces the expression of Haa1-regulated genes. We found that both pde2Δ and RAS2A18V19 reduce cell fitness in response to acetic acid stress, while ras2Δ increases cellular adaptation. There are three PKA catalytic subunits in yeast, encoded by TPK1, TPK2, and TPK3. We show that single mutations in TPK1 and TPK3 lead to the increased expression of Haa1-regulated genes, while tpk2Δ reduces their expression. Among tpk double mutations, tpk1Δ tpk3Δ greatly increases the expression of Haa1-regulated genes. We found that acetic acid stress in a tpk1Δ tpk3Δ double mutant induces a flocculation phenotype, which is reversed by haa1Δ. Our findings reveal PKA to be a negative regulator of the acetic acid stress response and may help engineer yeast strains with increased efficiency of bioethanol fermentation.

Thiamine pyrophosphokinase deficiency: report of two Chinese cases and a literature review.

Thiamine pyrophosphokinase (TPK) deficiency, is a rare autosomal recessive disorder of congenital metabolic dysfunction caused by variants in the TPK1 gene. TPK1 variants can lead to thiamine metabolic pathway obstacles, and its clinical manifestations are highly variable. We describe two cases of TPK deficiency with completely different phenotypes and different therapeutic effects, and 26 cases of previously reported were retrospectively reviewed to improve our understanding of the clinical and genetic features of the disease. Patients with TPK deficiency present with ataxia, dysarthria, dystonia, disturbance of consciousness, seizures, and other nervous system dysfunction. Different gene variant sites may lead to different clinical features and therapeutic effects. Gene analysis is important for the diagnosis of TPK deficiency caused by TPK1 variants, and thiamine supplementation has been the mainstay of treatment for TPK deficiency to date.

Effect of genetic variants in UBE2O and TPK1 on facial morphology of Koreans.

Human face is a highly heritable and complex trait. Many genome-wide analyses have identified genetic variants influencing facial morphology. Genome-wide association studies (GWASs) investigating facial morphologies of different populations provide a comprehensive insight into the genetic basis of the human face. Here, we report a GWAS of normal facial variation in Koreans using an array optimized for the Korean population (KoreanChip). We found that novel genetic variants encompassing four loci reached the genome-wide significance threshold. They include LOC107984547, UBE2O, TPK1, and LINC01148 loci associated with facial angle, brow ridge protrusion, nasal height, and eyelid curvature. Our results also validated previously published genetic loci, including FAT4, SOX9, and TBX3 loci. All confirmed genetic variants showed phenotypic differences involving each facial trait based on the effect of the minor allele. The present study highlights genetic signals associated with normal human facial variation and provides candidates for functional studies. Key points: GWAS of normal facial variation in the Korean population was conducted using a Korean genome chip.Previously reported genetic signals associated with FAT4, SOX9, and TBX3 loci were replicated in the Korean populations.Genetic signals in UBE2O and TPK1 loci were identified as novel variants for corresponding facial features.

Protein Kinase A Controls the Melanization of Candida auris through the Alteration of Cell Wall Components.

Candida auris, a multidrug-resistant fungal pathogen, significantly threatens global public health. Recent studies have identified melanin production, a key virulence factor in many pathogenic fungi that protects against external threats like reactive oxygen species, in C. auris. However, the melanin regulation mechanism remains elusive. This study explores the role of the Ras/cAMP/PKA signaling pathway in C. auris melanization. It reveals that the catalytic subunits Tpk1 and Tpk2 of protein kinase A (PKA) are essential, whereas Ras1, Gpr1, Gpa2, and Cyr1 are not. Under melanin-promoting conditions, the tpk1Δ tpk2Δ strain formed melanin granules in the supernatant akin to the wild-type strain but failed to adhere them properly to the cell wall. This discrepancy is likely due to a decreased expression of chitin-synthesis-related genes. Our findings also show that Tpk1 primarily drives melanization, with Tpk2 having a lesser impact. To corroborate this, we found that C. auris must deploy Tpk1-dependent melanin deposition as a defensive mechanism against antioxidant exposure. Moreover, we confirmed that deletion mutants of multicopper oxidase and ferroxidase genes, previously assumed to influence C. auris melanization, do not directly contribute to the process. Overall, this study sheds light on the role of PKA in C. auris melanization and enhances our understanding of the pathogenicity mechanisms of this emerging fungal pathogen.

Heat stress regulates the expression of TPK1 gene at transcriptional and post-transcriptional levels in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae cAMP regulates different cellular processes through PKA. The specificity of the response of the cAMP-PKA pathway is highly regulated. Here we address the mechanism through which the cAMP-PKA pathway mediates its response to heat shock and thermal adaptation in yeast. PKA holoenzyme is composed of a regulatory subunit dimer (Bcy1) and two catalytic subunits (Tpk1, Tpk2, or Tpk3). PKA subunits are differentially expressed under certain growth conditions. Here we demonstrate the increased abundance and half-life of TPK1 mRNA and the assembly of this mRNA in cytoplasmic foci during heat shock at 37 °C. The resistance of the foci to cycloheximide-induced disassembly along with the polysome profiling analysis suggest that TPK1 mRNA is impaired for entry into translation. TPK1 expression was also evaluated during a recurrent heat shock and thermal adaptation. Tpk1 protein level is significantly increased during the recovery periods. The crosstalk of cAMP-PKA pathway and CWI signalling was also studied. Wsc3 sensor and some components of the CWI pathway are necessary for the TPK1 expression upon heat shock. The assembly in foci upon thermal stress depends on Wsc3. Tpk1 expression is lower in a wsc3∆ mutant than in WT strain during thermal adaptation and thus the PKA levels are also lower. An increase in Tpk1 abundance in the PKA holoenzyme in response to heat shock is presented, suggesting that a recurrent stress enhanced the fitness for the coming favourable conditions. Therefore, the regulation of TPK1 expression by thermal stress contributes to the specificity of cAMP-PKA signalling.

TPK1 as a predictive marker for the anti-tumour effects of simvastatin in gastric cancer.

The potential anti-tumour role of statins has been reported in various cancer types, including gastric cancer (GC). However, there are no biomarkers to identify patients who may benefit most from this treatment. We tested the effects of statins on 8 GC cell lines. Genes differentially expressed in simvastatin-sensitive and -resistant cell lines were used to identify potential biomarkers of simvastatin sensitivity. Patient-derived cell lines were used to mimic in vivo conditions. In simvastatin-sensitive SNU-5 cells, the levels of the PARP and cleaved caspase-3 apoptosis markers increased upon exposure to simvastatin. The levels of the PARP and cleaved caspase-3 levels were unchanged by simvastatin exposure in simvastatin-resistant SNU-668 cells. The proportion of apoptotic cells was increased in SNU-5 cells but not in SNU-668 cells under the same drug exposure conditions. Comparison of differentially expressed genes (DEGs) in sensitive and resistant cell lines identified 31 genes potentially involved in the cellular response to simvastatin. We confirmed that RNA expression of the TPK1 DEG was significantly increased in simvastatin-sensitive cell lines. TPK1 knockdown in a simvastatin-sensitive GC SNU5 cell line, decreased the anti-tumour effects of simvastatin, while TPK1 overexpression enhanced the anti-tumour effect of simvastatin. Therefore, TPK1 expression can be used as a predictive marker of the anti-tumour effects of statin treatment in patients with cancer, especially in those with GC.

Whole Exome Sequencing Identifies a Novel Mutation of TPK1 in a Chinese Family with Recurrent Ataxia.

TPK deficiency, also known as thiamine metabolism dysfunction syndrome 5, is a rare autosomal recessive disorder of inborn error of metabolism caused by TPK1 gene mutation. Its clinical manifestation is highly variable, ranging from spontaneous remission to fatal metabolic crisis. Here, we describe two affected siblings in a Chinese family presenting with recurrent episodes of acute ataxia. Whole exome sequencing identified a homozygous missense variant c.382C > T (p.Leu128Phe) in the TPK gene, which is located in the thiamine binding domain and affects a highly conserved amino acid. Besides, a review of the 18 previously reported patients provides a better understanding of the clinical and genetic features of this disorder. TPK deficiency may be an under-diagnosed cause of acute encephalopathy and ataxia. Given the potential benefit of early intervention, TPK deficiency should be considered in patients with episodic encephalopathy or ataxia, especially those associated with lactic acidosis and α-ketoglutaric aciduria. Significant decreased TPP in the blood is a strong hint of the disease. WES (whole exome sequencing) can help to further identify the molecular diagnosis.

Eleven novel mutations and clinical characteristics in seven Chinese patients with thiamine metabolism dysfunction syndrome.

Thiamine metabolism dysfunction syndrome (THMD) comprises a group of clinically and genetically heterogeneous encephalopathies with autosomal recessive inheritance. Four genes, SLC19A3, SLC25A19, SLC19A2, and TPK1, are associated with this disorder. This study aimed to explore the clinical, biochemical and molecular characteristics of seven Chinese patients with THMD. Targeted next-generation sequencing of mitochondrial DNA and nuclear DNA was used to identify the causative mutations. The patients presented with subacute encephalopathy between the ages of 1-27 months. Brain magnetic resonance imaging (MRI) revealed abnormalities in the basal ganglia, indicating Leigh syndrome. Urine α-ketoglutarate in five patients was elevated. In four patients, five novel mutations (c.1276_1278delTAC, c.265A > C, c.197T > C, c.850T > C, whole gene deletion) were found in SLC19A3, which is associated with THMD2. In two patients, four novel mutations (c.194C > T, c.454C > A, c.481G > A, and c.550G > C) were identified in SLC25A19, supporting a diagnosis of THMD4. In one patient, two novel mutations (c.395T > C and c.614-1G > A) were detected in TPK1, which is indicative of THMD5. The patients received thiamine, biotin, and symptomatic therapy, upon which six patients demonstrated clinical improvement. Our findings expanded the phenotypic and genotypic spectrum of THMD, with eleven novel mutations identified in seven Chinese patients. Early diagnosis and treatment have a significant impact on prognosis.

Thiamine phosphokinase deficiency and mutation in TPK1 presenting as biotin responsive basal ganglia disease.

The product of thiamine phosphokinase is the cofactor for many enzymes, including the dehydrogenases of pyruvate, 2-ketoglutarate and branched chain ketoacids. Its deficiency has recently been described in a small number of patients, some of whom had a Leigh syndrome phenotype. The patient who also had a Leigh phenotype was initially found to have a low concentration of biotin in plasma and massive urinary excretion of biotin. Despite treatment with biotin and thiamine, her disease was progressive. Mutations c.311delG and c.426G > C were found in the TPK1 gene.

Identification of two novel TPK1 gene mutations in a Chinese patient with thiamine pyrophosphokinase deficiency undergoing whole exome sequencing.

Background The mutations of thiamine pyrophosphokinase-1 (TPK1) gene have been frequently studied in some patients with thiamine metabolism dysfunction syndrome-5 (THMD5), while TPK1 mutations in Chinese patients have been investigated by only homozygous. A search of the literature on the mutations in the Chinese population currently published revealed that no reports of compound heterozygous mutations were reported. Here, we report a Chinese patient with compound heterozygous TPK1 mutations who underwent magnetic resonance imaging (MRI), whole exome sequencing (WES), molecular diagnosis, bioinformatics analysis, and three-dimensional (3D) protein structure analysis. Case presentation A Chinese boy was born after an uneventful pregnancy to non-consanguineous and healthy parents. On the sixth day after his birth, the lactate level of the patient was between 8.6 mmol/L and 14.59 mmol/L in plasma (the normal level is in the range of 0.5-2.2 mmol/L). Lactate was reduced to the normal level after rehydration, acid correction, expansion, and other treatments. After 4 months, the patient presented with an acute, 3-h-long, non-induced convulsions, and was admitted to our hospital for weakness, decreased oral intake, and lethargy. Results achieved by electroencephalography (EEG), cerebrospinal fluid, and other biochemical findings were normal. A visible hemorrhagic lesion was also observed in the brain. Seizures increased significantly during infection, which was accompanied by higher lactic acid levels. MRI of the brain showed an obvious signal shadow, in which bilateral frontal and temporal parietal subarachnoid cavities were widened, and more abnormal signals were observed; therefore, further consideration of hypoxic-ischemic encephalopathy and genetic metabolic disease was taken into account. Conclusions The results of WES revealed that the patient was associated with compound heterozygous mutations NM_022445.3:c.[263G>A]; [226A>G] of TPK1. His parents were non-consanguineous; while his father was found to be a heterozygous carrier with the mutation c.[263G>A], his mother was identified as a heterozygous carrier with the mutation c.[226A>G]. The results indicated that the patient had a compound heterozygous TPK1 mutation, and this is the first reported case in China.

Utility of Whole Blood Thiamine Pyrophosphate Evaluation in TPK1-Related Diseases.

TPK1 mutations are a rare, but potentially treatable, cause of thiamine deficiency. Diagnosis is challenging given the phenotypic overlap that exists with other metabolic and neurological disorders. We report a case of TPK1-related disease presenting with Leigh-like syndrome and review the diagnostic utility of thiamine pyrophosphate (TPP) blood measurement. The proband, a 35-year-old male, presented at four months of age with recurrent episodes of post-infectious encephalopathy. He subsequently developed epilepsy, learning difficulties, sensorineural hearing loss, spasticity, and dysphagia. There was a positive family history for Leigh syndrome in an older brother. Plasma lactate was elevated (3.51 mmol/L) and brain MRI showed bilateral basal ganglia hyperintensities, indicative of Leigh syndrome. Histochemical and spectrophotometric analysis of mitochondrial respiratory chain complexes I, II+III, and IV was normal. Genetic analysis of muscle mitochondrial DNA was negative. Whole exome sequencing of the proband confirmed compound heterozygous variants in TPK1: c. 426G>C (p. Leu142Phe) and c. 258+1G>A (p.?). Blood TPP levels were reduced, providing functional evidence for the deleterious effects of the variants. We highlight the clinical and bioinformatics challenges to diagnosing rare genetic disorders and the continued utility of biochemical analyses, despite major advances in DNA sequencing technology, when investigating novel, potentially disease-causing, genetic variants. Blood TPP measurement represents a fast and cost-effective diagnostic tool in TPK1-related diseases.

Development of a high efficient promoter finding method based on transient transfection.

In metazoan genome, the mechanism of gene expression regulation between transcriptional regulatory elements and their target gene is spatiotemporal. Active promoters possess many specific chromosomal features, such as hypersensitive to DNaseI and enrichment of specific histone modifications. In this article, we proposed a novel method which possesses a high efficiency to find promoters in vitro. A promoter-trap library was constructed with totally 706 random mouse genomic DNA fragment clones, and 260 promoter-active fragments of the library were screened by transient transfection into 4T1 cells. To demonstrate the accuracy of this promoter finding method, 13 fragments with promoter activities were randomly selected for published DNase-seq and ChIP-seq data analysis, downstream transcripts prediction and expression confirmation. qRT-PCR results showed that six predicted transcription units were successfully amplified in different mouse tissues/cells or in reconstituted mouse mammary tumors. Our results indicate that this promoter finding method can successfully detect the promoter-active fragments and their downstream transcripts.