"Progressive myoclonic ataxia and developmental/epileptic encephalopathy associated with a novel homozygous mutation in TCN2 gene".

BACKGROUND: Transcobalamin II (TCN2) defect is a rare metabolic disorder associated with a range of neurological manifestations, including mild developmental delay, severe intellectual disability, ataxia, and, in some cases, seizures. Cobalamin, an essential nutrient, plays a crucial role in central nervous system myelination. CLINICAL PRESENTATION: We present a family with an index patient who exhibited progressive neurodevelopmental regression starting at 9 months of age, accompanied by myoclonic seizures, ataxia, and tremor. No significant hematological abnormalities were observed. Exome sequencing analysis identified a novel homozygous mutation, c.3G>A - P(Met1I), affecting the acceptor site of intron 4 of the TCN2 gene (chromosome 22: 31003321, NM_000355.4), leading to likely pathogenic variant potentially affecting translation. Following treatment with hydroxocobalamin, the patient demonstrated partial clinical improvement. He has a sibling with overt hematological abnormalities and subtle neurological abnormalities who is homozygous to the same mutation. Both parents are heterozygous for the same mutation. CONCLUSIONS: In infants presenting with unexplained non-specific neurological symptoms, irrespective of classical signs of vitamin B12 deficiency, evaluation for TCN2 defect should be considered. Early diagnosis and appropriate management can lead to favorable outcomes.

FTY720 requires vitamin B12-TCN2-CD320 signaling in astrocytes to reduce disease in an animal model of multiple sclerosis.

Vitamin B12 (B12) deficiency causes neurological manifestations resembling multiple sclerosis (MS); however, a molecular explanation for the similarity is unknown. FTY720 (fingolimod) is a sphingosine 1-phosphate (S1P) receptor modulator and sphingosine analog approved for MS therapy that can functionally antagonize S1P1. Here, we report that FTY720 suppresses neuroinflammation by functionally and physically regulating the B12 pathways. Genetic and pharmacological S1P1 inhibition upregulates a transcobalamin 2 (TCN2)-B12 receptor, CD320, in immediate-early astrocytes (ieAstrocytes; a c-Fos-activated astrocyte subset that tracks with experimental autoimmune encephalomyelitis [EAE] severity). CD320 is also reduced in MS plaques. Deficiency of CD320 or dietary B12 restriction worsens EAE and eliminates FTY720's efficacy while concomitantly downregulating type I interferon signaling. TCN2 functions as a chaperone for FTY720 and sphingosine, whose complex induces astrocytic CD320 internalization, suggesting a delivery mechanism of FTY720/sphingosine via the TCN2-CD320 pathway. Taken together, the B12-TCN2-CD320 pathway is essential for the mechanism of action of FTY720.

Reference intervals and stability of haptocorrin and holotranscobalamin in Danish children and elderly.

BACKGROUND: Haptocorrin (HC) and holotranscobalamin (holoTC) carry vitamin B12 (B12) in the circulation and can be useful biomarkers for evaluating B12 status. The concentration of both proteins depends on age, but data on reference intervals for children and the elderly are sparse. Similarly, not much is known about the effect of preanalytical factors. METHODS: HC plasma samples from healthy elderly > 65 years (n = 124) were analysed, and both HC and holoTC were analysed in paediatric serum samples ≤ 18 years (n = 400). Furthermore, we investigated assay precision and stability. RESULTS: HC and holoTC were effected by age. We established reference intervals for HC: 2-10 years, 369-1237 pmol/L; 11-18 years, 314-1128 pmol/L; 65-82 years, 242-680 pmol/L and for holoTC: 2-10 years, 46-206 pmol/L; 11-18 years, 30-178 pmol/L. Analytical coefficients of variations of 6.0-6.8% and 7.9-15.7% were found for HC and holoTC, respectively. HC were affected when stored at room temperature and by freeze/thaw. HoloTC was stable at room temperature and after delayed centrifugation. CONCLUSION: We present novel 95% age-related reference limits for HC and HoloTC in children, and for HC both in children and elderly. Moreover, we found HoloTC to be fairly stable when stored, whereas HC was more vulnerable to preanalytical factors.

Significance of anti-transcobalamin receptor antibodies in cutaneous arteritis revealed by proteome-wide autoantibody screening.

Cutaneous arteritis (CA) is a single-organ vasculitis that exclusively affects the small to medium-sized arteries of the skin. Diagnosis depends on a histological investigation with skin biopsy, which could be burdensome for both patients and clinicians. Moreover, the pathogenesis of CA remains unstudied, and treatment has not yet been established. Herein, we applied our proteome-wide autoantibody screening method to explore autoantibodies in the serum of CA patients. As a result, anti-transcobalamin receptor (TCblR) antibodies (Abs) were specifically detected in 24% of CA patients. Patients with positive anti-TCblR Abs were spared from peripheral neuropathy compared to those with negative anti-TCblR Abs, showing characteristics as CA confined to the skin. In addition, we revealed that anti-TCblR Abs trigger the autocrine loop of interleukin-6 mediated by tripartite motif-containing protein 21 in human endothelial cells and induce periarterial inflammation in murine skin. Furthermore, we demonstrated that methylcobalamin, a ligand of TCblR, ameliorates inflammation caused by anti-TCblR Abs both in vitro and in vivo. Collectively, our investigation unveils the pathologic significance of anti-TCblR Abs in CA and their potential as a diagnostic marker and a pathophysiology-oriented therapeutic target.

Vitamin B12 binding to mutated human transcobalamin, in-silico study of TCN2 alanine scanning and ClinVar missense mutations/SNPs.

Many missense mutations/SNPs of the TCN2 gene (which yield Transcobalamin (TC)) were reported in the literature but no study is available about their effect on binding to vitamin B12(B12) at the structural level experimentally nor computationally. Predict the effect of TC missense mutations/SNPs on binding affinity to B12 and characterize their contacts to B12 at the structural level. TC-B12 binding energy difference from the wildtype (ΔΔGmut) was calculated for 378 alanine scanning mutations and 76 ClinVar missense mutations, repeated on two distinct X-ray structures of holoTC namely 2BB5 and 4ZRP. Destabilizing mutations then went through 100 ns molecular dynamics simulation to study their effect on TC-B12 binding at the structural level employing 2BB5 structure. Out of the studied 454 mutations (378 alanine mutations + 76 ClinVar mutations), 19 were destabilizing representing 17 amino acid locations. Mutation energy results show a neutral effect on B12 binding of several missense SNPs reported in the literature including I23V, G94S, R215W, P259R, S348F, L376S, and R399Q. Compared to the wildtype, all the destabilizing mutations have higher average RMSD-Ligand in the last 25% of the MD simulation trajectories and lower average hydrogen bond count while the other parameters vary. Previously reported TCN2 SNPs with an unknown effect on TC-B12 binding were found to have a neutral effect in the current study based on mutation energy calculations. Also, we reported 17 possible amino acids that destabilize TC-B12 binding upon mutation (four listed in ClinVar) and studied their structural effect computationally.

Residues L55 and W69 of Tva Mediate Entry of Subgroup A Avian Leukosis Virus.

The receptor of the subgroup A avian leukosis virus (ALV-A) in chicken is Tva, which is the homologous protein of human CD320 (huCD320), contains a low-density lipoprotein (LDL-A) module and is involved in the uptake of transcobalamin bound vitamin B12/cobalamin (Cbl). To map the functional determinants of Tva responsible for ALV-A receptor activity, a series of chimeric receptors were created by swapping the LDL-A module fragments between huCD320 and Tva. These chimeric receptors were then used for virus entry and binding assays to map the minimal ALV-A functional domain of Tva. The results showed that Tva residues 49 to 71 constituted the minimal functional domain that directly interacted with the ALV-A gp85 protein to mediate ALV-A entry. Single-residue substitution analysis revealed that L55 and W69, which were spatially adjacent on the surface of the Tva structure, were key residues that mediate ALV-A entry. Structural alignment results indicated that L55 and W69 substitutions did not affect the Tva protein structure but abolished the interaction force between Tva and gp85. Furthermore, substituting the corresponding residues of huCD320 with L55 and W69 of Tva converted huCD320 into a functional receptor of ALV-A. Importantly, soluble huCD320 harboring Tva L55 and W69 blocked ALV-A entry. Finally, we constructed a Tva gene-edited cell line with L55R and W69L substitutions that could fully resist ALV-A entry, while Cbl uptake was not affected. Collectively, our findings suggested that amino acids L55 and W69 of Tva were key for mediating virus entry. IMPORTANCE Retroviruses bind to cellular receptors through their envelope proteins, which is a crucial step in infection. While most retroviruses require two receptors for entry, ALV-A requires only one. Various Tva alleles conferring resistance to ALV-A, including Tvar1 (C40W substitution), Tvar2 (frame-shifting four-nucleotide insertion), Tvar3, Tvar4, Tvar5, and Tvar6 (deletion in the first intron), are known. However, the detailed entry mechanism of ALV-A in chickens remains to be explored. We demonstrated that Tva residues L55 and W69 were key for ALV-A entry and were important for correct interaction with ALV-A gp85. Soluble Tva and huCD320 harboring the Tva residues L55 and W69 effectively blocked ALV-A infection. Additionally, we constructed gene-edited cell lines targeting these two amino acids, which completely restricted ALV-A entry without affecting Cbl uptake. These findings contribute to a better understanding of the infection mechanism of ALV-A and provided novel insights into the prevention and control of ALV-A.

Diagnostic and Therapeutic Perspectives Associated to Cobalamin-Dependent Metabolism and Transcobalamins' Synthesis in Solid Cancers.

Cobalamin or vitamin B12 (B12) is a cofactor for methionine synthase and methylmalonyl-CoA mutase, two enzymes implicated in key pathways for cell proliferation: methylation, purine synthesis, succinylation and ATP production. Ensuring these functions in cancer cells therefore requires important cobalamin needs and its uptake through the transcobalamin II receptor (TCII-R). Thus, both the TCII-R and the cobalamin-dependent metabolic pathways constitute promising therapeutic targets to inhibit cancer development. However, the link between cobalamin and solid cancers is not limited to cellular metabolism, as it also involves the circulating transcobalamins I and II (TCI or haptocorrin and TCII) carrier proteins, encoded by TCN1 and TCN2, respectively. In this respect, elevations of B12, TCI and TCII concentrations in plasma are associated with cancer onset and relapse, and with the presence of metastases and worse prognosis. In addition, TCN1 and TCN2 overexpressions are associated with chemoresistance and a proliferative phenotype, respectively. Here we review the involvement of cobalamin and transcobalamins in cancer diagnosis and prognosis, and as potential therapeutic targets. We further detail the relationship between cobalamin-dependent metabolic pathways in cancer cells and the transcobalamins' abundancies in plasma and tumors, to ultimately hypothesize screening and therapeutic strategies linking these aspects.

Probing the functional consequence and clinical relevance of CD320 p.E88del, a variant in the transcobalamin receptor gene.

The biological and clinical significance of the p.E88del variant in the transcobalamin receptor, CD320, is unknown. This allele is annotated in ClinVar as likely benign, pathogenic, and of uncertain significance. To determine functional consequence and clinical relevance of this allele, we employed cell culture and genetic association studies. Fibroblasts from 16 CD320 p.E88del homozygotes exhibited reduced binding and uptake of cobalamin. Complete ascertainment of newborns with transiently elevated C3 (propionylcarnitine) in New York State demonstrated that homozygosity for CD320 p.E88del was over-represented (7/348, p < 6 × 10-5 ). Using population data, we estimate that ~85% of the p.E88del homozygotes born in the same period did not have elevated C3, suggesting that cobalamin metabolism in the majority of these infants with this genotype is unaffected. Clinical follow-up of 4/9 homozygous individuals uncovered neuropsychological findings, mostly in speech and language development. None of these nine individuals exhibited perturbation of cobalamin metabolism beyond the newborn stage even during periods of acute illness. Newborns homozygous for this allele in the absence of other factors are at low risk of requiring clinical intervention, although more studies are required to clarify the natural history of various CD320 variants across patient populations.

Generation of nanobodies targeting the human, transcobalamin-mediated vitamin B12 uptake route.

Cellular uptake of vitamin B12 in humans is mediated by the endocytosis of the B12 carrier protein transcobalamin (TC) via its cognate cell surface receptor TCblR, encoded by the CD320 gene. Because CD320 expression is associated with the cell cycle and upregulated in highly proliferating cells including cancer cells, this uptake route is a potential target for cancer therapy. We developed and characterized four camelid nanobodies that bind holo-TC (TC in complex with B12 ) or the interface of the human holo-TC:TCblR complex with nanomolar affinities. We determined X-ray crystal structures of these nanobodies bound to holo-TC:TCblR, which enabled us to map their binding epitopes. When conjugated to the model toxin saporin, three of our nanobodies caused growth inhibition of HEK293T cells and therefore have the potential to inhibit the growth of human cancer cells. We visualized the cellular binding and endocytic uptake of the most potent nanobody (TC-Nb4) using fluorescent light microscopy. The co-crystal structure of holo-TC:TCblR with another nanobody (TC-Nb34) revealed novel features of the interface of TC and the LDLR-A1 domain of TCblR, rationalizing the decrease in the affinity of TC-B12 binding caused by the Δ88 mutation in CD320.

One-carbon metabolism and global DNA methylation in mothers of individuals with Down syndrome.

Down syndrome (DS) is the most common chromosomal disorder, resulting from the failure of normal chromosome 21 segregation. Studies have suggested that impairments within the one-carbon metabolic pathway can be of relevance for the global genome instability observed in mothers of individuals with DS. Based on the association between global DNA hypomethylation, genome instability, and impairments within the one-carbon metabolic pathway, the present study aimed to identify possible predictors, within the one-carbon metabolism, of global DNA methylation, measured by methylation patterns of LINE-1 and Alu repetitive sequences, in mothers of individuals with DS and mothers of individuals without the syndrome. In addition, we investigated one-carbon genetic polymorphisms and metabolites as maternal predisposing factors for the occurrence of trisomy 21 in children. Eighty-three samples of mothers of children with DS with karyotypically confirmed free trisomy 21 (case group) and 84 of mothers who had at least one child without DS or any other aneuploidy were included in the study. Pyrosequencing assays were performed to access global methylation. The results showed that group affiliation (case or control), betaine-homocysteine methyltransferase (BHMT) G742A and transcobalamin 2 (TCN2) C776G polymorphisms, and folate concentration were identified as predictors of global Alu DNA methylation values. In addition, thymidylate synthase (TYMS) 28-bp repeats 2R/3R or 3R/3R genotypes are independent maternal predisposing factors for having a child with DS. This study adds evidence that supports the association of impairments in the one-carbon metabolism, global DNA methylation, and the possibility of having a child with DS.

Vitamin B12 Deficiency Dysregulates m6A mRNA Methylation of Genes Involved in Neurological Functions.

INTRODUCTION: Vitamin B12 deficiency presents various neurological manifestations, such as cognitive dysfunction, mental retardation, or memory impairment. However, the involved molecular mechanisms remain to date unclear. Vitamin B12 is essential for synthesizing S-adenosyl methionine (SAM), the methyl group donor used for almost all transmethylation reactions. Here, we investigate the m6A methylation of mRNAs and their related gene expression in models of vitamin B12 deficiency. METHODS AND RESULTS: This study observes two cellular models deficient in vitamin B12 and hippocampi of mice knock-out for the CD320 receptor. The decrease in SAM levels resulting from vitamin B12 deficiency is associated with m6 A reduced levels in mRNAs. This is also potentially mediated by the overexpression of the eraser FTO. We further investigate mRNA methylation of some genes involved in neurological functions targeted by the m6A reader YTH proteins. We notably observe a m6A hypermethylation of Prkca mRNA and a consistently increased expression of PKCα, a kinase involved in brain development and neuroplasticity, in the two cellular models. CONCLUSION: Our data show that m6A methylation in mRNA could be one of the contributing mechanisms that underlie the neurological manifestations produced by vitamin B12 deficiency.

Elevated holo-transcobalamin in Gaucher disease type II: A case report.

Gaucher disease (GD), one of the most common lysosomal disorders, is caused by deficiency of ß-glucocerebrosidase. Based on the presence and severity of neurological complications, GD is classified into types I, II (the most severe form), and III. Abnormalities in systemic markers of vitamin B12 (B12 ) metabolism have been reported in GD type I patients, suggesting a higher prevalence of B12 deficiency in these patients. A 2-month-old male with GD type II was admitted to the hospital presenting jaundice, hepatosplenomegaly, and ichthyosis. At admission, cholestasis and ascites, abnormal liver function enzymes, prolonged prothrombin time, and high levels of B12 were confirmed. Analysis of biomarkers of B12 status revealed elevated B12 and holo-transcobalamin (holo-TC) levels. The B12 profile found in our patient is the opposite to what is described for GD type I patients. Holo-TC may increase in inflammatory states or due to liver diseases. In GD, the accumulation of glucocerebroside may be a trigger that initiates a systemic inflammatory reaction, characterized by macrophage activation. We suggest higher levels of holo-TC could be associated with a more severe (neuronopathic) GD, and be a biomarker of GD type II.

Intracellular and Tissue Levels of Vitamin B12 in Hepatocytes Are Modulated by CD320 Receptor and TCN2 Transporter.

The liver mass constitutes hepatocytes expressing receptors for vitamin B12 (B12)-bound transporters in circulation. However, intrahepatic and circulating B12 interrelationship levels remain unclear. We assessed the intracellular B12 levels at various circulating B12 concentrations in human HepG2 cell-line and liver tissue levels of B12 in the C57BL/6 mouse model. In HepG2 cells treated with a range of B12 concentrations, the intracellular and circulatory B12 levels, transcript and protein levels of B12 receptor (CD320) and transporter (TCN2) were determined using immunoassays, qRT-PCR and Western blot, respectively. Similar assessments were done in plasma and liver tissue of C57BL/6 mice, previously fed a diet of either a high or low B12 (30.82 µg B12/kg and 7.49 µg B12/kg, respectively) for 8-10 weeks. The physiological B12 status (0.15-1 nM) resulted in increased levels of intracellular B12 in HepG2 cells compared to supraphysiological levels of B12 (>1 nM). Gene and protein expression of CD320 and TCN2 were also higher at physiological levels of B12. Progressively increasing extracellular B12 to supraphysiological levels led to relative decreased levels of intracellular B12, lower expression of gene and protein levels of CD320 and TCN2. Similar results were observed in liver tissue from mice fed on a low B12 diet verses high B12 diet. These findings suggest that unlike supraphysiological B12, physiological levels of B12 in the extracellular media or circulation accelerates active transport of B12, and expression of CD320 and TCN2, resulting in higher relative uptake of B12 in hepatocytes.

Cross-sectional association between vitamin B12 status and probable postpartum depression in Indian women.

BACKGROUND: Vitamin B12 is an essential micronutrient for neurological function, as it leads to the regeneration of methionine from homocysteine, which is precursor of biologically active molecule S-Adenosyl Methionine (SAM). Pregnancy is a state of increased demand and delayed postpartum repletion of nutrients may predispose women to depression. METHODS: We included women who visited the hospital at 6-weeks postpartum for a regular checkup. Inclusion criteria were age (18-50 years), and willingness to donate venous sample for analysis. Exclusion criteria included previous history of mood disorders or antidepressant medication use, and any systemic illness like hypothyroidism, epilepsy, diabetes, and hypertension. Based on EPDS score of 10 as a cutoff, 217 women with probable postpartum depression (PPD) and equal number of age and BMI matched controls were included. Plasma total vitamin B12, holotranscobalamin (holotc), homocysteine (hcy), methyl malonic acid (MMA), 5-methyl tetrahydrofolate (THF), SAM and serotonin levels were estimated using commercially available ELISA kits. Combined B12 (cB12) score was calculated from study parameters. Multivariate analysis was performed to assess the risk of probable postpartum depression. RESULTS: Total vitamin B12 and combined B12 score were found to be significantly lower (p = 0.001) and MMA (p = 0.002) and 5-methyl THF (p < 0.001) levels were higher in women with probable depression than women without probable PPD. Women in the lowest vitamin B12 quartile had 4.53 times higher likelihood of probable postpartum depression (p < 0.001). Multivariate analysis demonstrated that decreasing vitamin B12 (OR = 0.394; 95% CI: 0.189-0.822) and cB12 (OR = 0.293; 95% CI: 0182-0.470) and increasing MMA (OR = 2.14; 95% CI: 1.63-2.83) and 5-methyl THF levels (OR = 3.29; 95% CI: 1.59-6.83) were significantly associated with the risk of probable PPD. CONCLUSION: Low vitamin B12 may contribute to depressive symptoms in vulnerable postpartum period.

Associations of Serum Folate and Holotranscobalamin with Cardiometabolic Risk Factors in Rural and Urban Cameroon.

A low intake of fruit and vegetables and a high intake of meat are associated with higher cardiometabolic disease risk; however much prior research has relied on subjective methods for dietary assessment and focused on Western populations. We aimed to investigate the association of blood folate as an objective marker of fruit and vegetable intake and holotranscobalamin (holoTC) as a marker of animal-sourced food intake with cardiometabolic risk factors. We conducted a population-based cross-sectional study on 578 adults (mean ± SD age = 38.2 ± 8.6 years; 64% women). The primary outcome was a continuous metabolic syndrome score. The median serum folate was 12.9 (IQR: 8.6-20.5) nmol/L and the mean holoTC was 75 (SD: 34.3) pmol/L. Rural residents demonstrated higher serum folate concentrations (15.9 (9.8-25.9) nmol/L) than urban residents (11.3 (7.9-15.8) nmol/L), but lower holoTC concentrations (rural: 69.8 (32.9) pmol/L; urban: 79.8 (34.9)) pmol/L, p < 0.001 for both comparisons. There was an inverse association between serum folate and metabolic syndrome score by -0.20 in the z-score (95% CI, -0.38 to -0.02) per 10.8 (1 SD) of folate) in a model adjusted for socio-demographic factors, smoking status, alcohol intake, BMI, and physical activity. HoloTC was positively associated with the metabolic syndrome score in unadjusted analysis (0.33 (95% CI, 0.10 to 0.56)) but became non-significant (0.17 (-0.05 to 0.39)) after adjusting for socio-demographic and behavioural characteristics. In conclusion, serum folate and holoTC were associated with the metabolic syndrome score in opposite directions. The positive association between serum holoTC and the metabolic syndrome score was partly dependent on sociodemographic characteristics. These findings suggest that, based on these biomarkers reflecting dietary intakes, public health approaches promoting a higher intake of fruit and vegetables may lower cardiometabolic risk factors in this population.

Cellular uptake of vitamin B12: Role and fate of TCblR/CD320, the transcobalamin receptor.

Cellular uptake of vitamin B12 (cobalamin, Cbl) is mediated by a cell surface receptor (TCblR/CD320) that binds transcobalamin (TC) saturated with Cbl. TC is secreted by the vascular endothelium, has a relatively short half-life, binds Cbl with high affinity and presents the vitamin to the receptor for cellular uptake. Here we show binding and internalization of the TC-Cbl complex along with its' receptor (TCblR) in several human cell lines. The expression of TCblR is linked to the cell cycle with highest expression in actively proliferating cells. Upon binding TC-Cbl, the receptors appear to segregate on the plasma membrane and are internalized over the course of 30-60 min. Subsequently, the receptors appear to be destroyed along with the TC, which results in the release of free Cbl in the lysosome. The appearance of TCblR on the cell surface is limited to newly synthesized protein without contribution from recycling of the receptor. Therefore, Cbl uptake into cells is fully dependent on the expression of newly synthesized TCblR that is up-regulated in actively proliferating cells. The cell cycle-associated up-regulation of TCblR in cancers provides a route for targeted drug delivery.

Clinicopathological Analysis and Prognostic Assessment of Transcobalamin I (TCN1) in Patients with Colorectal Tumors.

BACKGROUND Colorectal cancer (CRC) is one of the most common malignancies worldwide. Overall survival (OS) of patients is largely dependent on disease stage at diagnosis and/or surgical resection. TCN1 mainly encodes the vitamin B12 transporter, transcobalamin. Early studies show that TCN1 is a marker of CRC progression, but the impact of TCN1 on survival is unclear. MATERIAL AND METHODS We reviewed and analyzed colorectal tumor records, summarized the clinicopathological data, performed immunohistochemical detection of TCN1 again, and semi-quantitatively analyzed protein expression in tumor tissue, non-tumor tissue, and lymph nodes. We followed up patients for 5-year survival. RESULTS Of 123 patients, 60 (48.7%) had a strong TCN1 immunohistochemical reaction, 36 (29.3%) had a moderate immune response, and 27 (22.0%) had weak expression. The level of immunohistochemical reactivity of TCN1 was correlated with the degree of histological differentiation (H (2.92)=4.976; P=0.083). Survival analysis showed that OS in patients with low TCN1 expression was significantly longer than that in the medium and high TCN1 expression groups (P=0.045). Five-year OS in patients with low, medium, and high TCN1 expression was 88.9%, 50.0%, and 40.0%, respectively. In univariate analysis, TCN1 immune expression was significantly correlated with the 5-year survival rate. CONCLUSIONS Although independent risk factors affecting survival of patients with CRC are age, serum CA125, CA19-9, lymph node metastasis, and nerve invasion, negative factors affecting overall 5-year survival in TCN1 should not be ignored, because its high expression suggests a worse clinical prognosis.

High expression of TCN1 is a negative prognostic biomarker and can predict neoadjuvant chemosensitivity of colon cancer.

Transcobalamin (TCN1) is a vitamin B12 (cobalamin)-binding protein that regulates cobalamin homeostasis. Recent studies and bioinformatic analyses have found that TCN1 is highly expressed in cancer tissues and is associated with tumour aggressiveness and poor prognosis. The present study aimed to detect TCN1 as a novel biomarker for prognosis and chemosensitivity of colon cancer. Next-generation sequencing showed that TCN1 was one of several upregulated mRNAs in colon cancer, which was verified by further bioinformatics analyses. Western blotting (n = 9) and quantitative real time polymerase chain reaction (qRT-PCR, n = 30) revealed that TCN1 was highly expressed in colon cancer tissues at both the protein and mRNA level. A total of 194 cases of colon cancer were examined by immunohistochemistry and revealed that TCN1 expression level was related to advanced stages (P < 0.005). Kaplan-Meier analysis verified that patients with lower TCN1 expression usually had longer overall survival (P = 0.008). In addition, TCN1 was highly expressed in pulmonary metastatic tumour tissues (n = 37, P = 0.025) and exhibited higher levels in right-sided colon cancer than in left-sided colon cancer (P = 0.029). TCN1 expression in specimens that had received neoadjuvant chemotherapy decreased compared with that in colonoscopy biopsy tissues (n = 42, P = 0.009). Further bioinformatics analyses verified that apoptosis pathways might have a role in high TCN1 expression. All the studies revealed that TCN1 expression in colon cancer was significantly associated with malignant biological behaviour. Therefore, TCN1 could be used as a novel biomarker for colon cancer aggressiveness and prognosis and might also be a potential biomarker for predicting neoadjuvant chemosensitivity.

Diagnostic Accuracy of Holotranscobalamin, Vitamin B12, Methylmalonic Acid, and Homocysteine in Detecting B12 Deficiency in a Large, Mixed Patient Population.

Four biomarkers are commonly employed to diagnose B12 deficiency: vitamin B12 (B12), holotranscobalamin (HoloTC), methylmalonic acid (MMA), and homocysteine (Hcy). 4cB12, a combined index of the B12 status, has been suggested to improve the recognition of B12 deficiency. We aimed to evaluate the four different markers for detecting B12 deficiency, as determined by 4cB12. Within a large, mixed patient population, 11,833 samples had concurrent measurements of B12, HoloTC, MMA, and Hcy. 4cB12 was calculated according to the methods described by Fedosov. Diagnostic cutoffs as well as diagnostic accuracy for the detection of B12 deficiency were assessed with receiver operating characteristic (ROC) analysis. The median age was 56 years, and women accounted for 58.8% of the samples. Overall, the area under the curve (AUC) for the detection of subclinical B12 deficiency was highest for HoloTC (0.92), followed by MMA (0.91), B12 (0.9) and Hcy (0.78). The difference between HoloTC and B12 was driven by a significantly higher AUC for HoloTC (0.93) than for B12 (0.89), MMA (0.91), and Hcy in women 50 years and older (0.79; p < 0.05 for all). In the detection of subclinical B12 deficiency, there were no significant differences in the AUCs of HoloTC, B12, and MMA among men and women <50 years. In conclusion, in women < 50 years and in men, HoloTC, MMA, or Hcy do not appear superior to B12 for the detection of B12 deficiency. For women 50 years and older, HoloTC seems to be the preferred first-line marker for the detection of subclinical B12 deficiency.