Pyridoxal Phosphate Deficiency Research Articles

Vitamin B6 deficiency cooperates with oncogenic Ras to induce malignant tumors in Drosophila

Vitamin B6 is a water-soluble vitamin which possesses antioxidant properties. Its catalytically active form, pyridoxal 5’-phosphate (PLP), is a crucial cofactor for DNA and amino acid metabolism. The inverse correlation between vitamin B6 and cancer risk has been observed in several studies, although dietary vitamin B6 intake sometimes failed to confirm this association. However, the molecular link between vitamin B6 and cancer remains elusive. Previous work has shown that vitamin B6 deficiency causes chromosome aberrations (CABs) in Drosophila and human cells, suggesting that genome instability may correlate the lack of this vitamin to cancer. Here we provide evidence in support of this hypothesis. Firstly, we show that PLP deficiency, induced by the PLP antagonists 4-deoxypyridoxine (4DP) or ginkgotoxin (GT), promoted tumorigenesis in eye larval discs transforming benign RasV12 tumors into aggressive forms. In contrast, PLP supplementation reduced the development of tumors. We also show that low PLP levels, induced by 4DP or by silencing the sgllPNPO gene involved in PLP biosynthesis, worsened the tumor phenotype in another Drosophila cancer model generated by concomitantly activating RasV12 and downregulating Discs-large (Dlg) gene. Moreover, we found that RasV12 eye discs from larvae reared on 4DP displayed CABs, reactive oxygen species (ROS) and low catalytic activity of serine hydroxymethyltransferase (SHMT), a PLP-dependent enzyme involved in thymidylate (dTMP) biosynthesis, in turn required for DNA replication and repair. Feeding RasV12 4DP-fed larvae with PLP or ascorbic acid (AA) plus dTMP, rescued both CABs and tumors. The same effect was produced by overexpressing catalase in RasV12DlgRNAi 4DP-fed larvae, thus allowing to establish a relationship between PLP deficiency, CABs, and cancer. Overall, our data provide the first in vivo demonstration that PLP deficiency can impact on cancer by increasing genome instability, which is in turn mediated by ROS and reduced dTMP levels.

A gene-nutrient interaction between vitamin B6 and serine hydroxymethyltransferase (SHMT) affects genome integrity in Drosophila.

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, participates as a cofactor to one carbon (1C) pathway that produces precursors for DNA metabolism. The concerted action of PLP-dependent serine hydroxymethyltransferase (SHMT) and thymidylate synthase (TS) leads to the biosynthesis of thymidylate (dTMP), which plays an essential function in DNA synthesis and repair. PLP deficiency causes chromosome aberrations (CABs) in Drosophila and human cells, rising the hypothesis that an altered 1C metabolism may be involved. To test this hypothesis, we used Drosophila as a model system and found, firstly, that in PLP deficient larvae SHMT activity is reduced by 40%. Second, we found that RNAi-induced SHMT depletion causes chromosome damage rescued by PLP supplementation and strongly exacerbated by PLP depletion. RNAi-induced TS depletion causes severe chromosome damage, but this is only slightly enhanced by PLP depletion. dTMP supplementation rescues CABs in both PLP-deficient and PLP-proficient SHMTRNAi . Altogether these data suggest that a reduction of SHMT activity caused by PLP deficiency contributes to chromosome damage by reducing dTMP biosynthesis. In addition, our work brings to light a gene-nutrient interaction between SHMT decreased activity and PLP deficiency impacting on genome stability that may be translated to humans.

Vitamin B6 Deficiency Promotes Loss of Heterozygosity (LOH) at the Drosophila warts (wts) Locus

The active form of vitamin B6, pyridoxal 5′-phosphate (PLP), is a cofactor for more than 200 enzymes involved in many metabolic pathways. Moreover, PLP has antioxidant properties and quenches the reactive oxygen species (ROS). Accordingly, PLP deficiency causes chromosome aberrations in Drosophila, yeast, and human cells. In this work, we investigated whether PLP depletion can also cause loss of heterozygosity (LOH) of the tumor suppressor warts (wts) in Drosophila. LOH is usually initiated by DNA breakage in heterozygous cells for a tumor suppressor mutation and can contribute to oncogenesis inducing the loss of the wild-type allele. LOH at the wts locus results in epithelial wts homozygous tumors easily detectable on adult fly cuticle. Here, we found that PLP depletion, induced by two PLP inhibitors, promotes LOH of wts locus producing significant frequencies of wts tumors (~7% vs. 2.3%). In addition, we identified the mitotic recombination as a possible mechanism through which PLP deficiency induces LOH. Moreover, LOH of wts locus, induced by PLP inhibitors, was rescued by PLP supplementation. These data further confirm the role of PLP in genome integrity maintenance and indicate that vitamin B6 deficiency may impact on cancer also by promoting LOH.

Disorders affecting vitamin B6 metabolism.

Vitamin B6 is present in our diet in many forms, however, only pyridoxal 5'-phosphate (PLP) can function as a cofactor for enzymes. The intestine absorbs nonphosphorylated B6 vitamers, which are converted by specific enzymes to the active PLP form. The role of PLP is enabled by its reactive aldehyde group. Pathways reliant on PLP include amino acid and neurotransmitter metabolism, folate and 1-carbon metabolism, protein and polyamine synthesis, carbohydrate and lipid metabolism, mitochondrial function and erythropoiesis. Besides the role of PLP as a cofactor B6 vitamers also play other cellular roles, for example, as antioxidants, modifying expression and action of steroid hormone receptors, affecting immune function, as chaperones and as an antagonist of Adenosine-5'-triphosphate (ATP) at P2 purinoceptors. Because of the vital role of PLP in neurotransmitter metabolism, particularly synthesis of the inhibitory transmitter γ-aminobutyric acid, it is not surprising that various inborn errors leading to PLP deficiency manifest as B6 -responsive epilepsy, usually of early onset. This includes pyridox(am)ine phosphate oxidase deficiency (a disorder affecting PLP synthesis and recycling), disorders affecting PLP import into the brain (hypophosphatasia and glycosylphosphatidylinositol anchor synthesis defects), a disorder of an intracellular PLP-binding protein (PLPBP, previously named PROSC) and disorders where metabolites accumulate that inactivate PLP, for example, ALDH7A1 deficiency and hyperprolinaemia type II. Patients with these disorders can show rapid control of seizures in response to either pyridoxine and/or PLP with a lifelong dependency on supraphysiological vitamin B6 supply. The clinical and biochemical features of disorders leading to B6 -responsive seizures and the treatment of these disorders are described in this review.

The Relationship Between Vitamin B6, Diabetes and Cancer.

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, works as cofactor in numerous enzymatic reactions and it behaves as antioxidant molecule. PLP deficiency has been associated to many human pathologies including cancer and diabetes and the mechanism behind this connection is now becoming clearer. Inadequate intake of this vitamin increases the risk of many cancers; furthermore, PLP deprivation impairs insulin secretion in rats, whereas PLP supplementation prevents diabetic complications and improves gestational diabetes. Growing evidence shows that diabetes and cancer are correlated not only because they share same risk factors but also because diabetic patients have a higher risk of developing tumors, although the underlying mechanisms remain elusive. In this review, we will explore data obtained in Drosophila revealing the existence of a connection between vitamin B6, DNA damage and diabetes, as flies in the past decade turned out to be a promising model also for metabolic diseases including diabetes. We will focus on recent studies that revealed a specific role for PLP in maintaining chromosome integrity and glucose homeostasis, and we will show that these aspects are correlated. In addition, we will discuss recent data identifying PLP as a putative linking factor between diabetes and cancer.

Protective role of vitamin B6 (PLP) against DNA damage in Drosophila models of type 2 diabetes

Growing evidence shows that improper intake of vitamin B6 increases cancer risk and several studies indicate that diabetic patients have a higher risk of developing tumors. We previously demonstrated that in Drosophila the deficiency of Pyridoxal 5′ phosphate (PLP), the active form of vitamin B6, causes chromosome aberrations (CABs), one of cancer prerequisites, and increases hemolymph glucose content. Starting from these data we asked if it was possible to provide a link between the aforementioned studies. Thus, we tested the effect of low PLP levels on DNA integrity in diabetic cells. To this aim we generated two Drosophila models of type 2 diabetes, the first by impairing insulin signaling and the second by rearing flies in high sugar diet. We showed that glucose treatment induced CABs in diabetic individuals but not in controls. More interestingly, PLP deficiency caused high frequencies of CABs in both diabetic models demonstrating that hyperglycemia, combined to reduced PLP level, impairs DNA integrity. PLP-depleted diabetic cells accumulated Advanced Glycation End products (AGEs) that largely contribute to CABs as α-lipoic acid, an AGE inhibitor, rescued not only AGEs but also CABs. These data, extrapolated to humans, indicate that low PLP levels, impacting on DNA integrity, may be considered one of the possible links between diabetes and cancer.

Pyridoxal Phosphate Supplementation in Neuropediatric Disorders

Pyridoxal phosphate (PLP) is the active form of vitamin B6 and a cofactor in many enzyme reactions including neurotransmitter metabolism. PLP metabolism disturbances may mostly lead to refractory seizures. In this report, we review the main pathophysiological factors related with PLP deficiency and our experience in PLP treatment in pediatric patients with low-normal cerebrospinal fluid PLP values who presented epilepsy. Only one case had a definite diagnosis (Phelan-McDermid syndrome). The results of extensive metabolic workups and targeted genetic studies were normal for all patients. In 5 cases, the response to PLP supplementation (10-30mg/kg/d) was initially positive. PLP adverse reactions were noticed in 4 patients and PLP was discontinued; however, one of the most noticeable symptoms was an asymptomatic increase in liver enzymes. These negative results with PLP supplementation are worth reporting, to improve the information we use to treat our patients.

Carnosine Content in Skeletal Muscle Is Dependent on Vitamin B6 Status in Rats.

Carnosine, a histidine-containing dipeptide, is well known to be associated with skeletal muscle performance. However, there is limited information on the effect of dietary micronutrients on muscle carnosine level. Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, is involved in amino acid metabolisms in the body as a cofactor. We hypothesized that enzymes involved in β-alanine biosynthesis, the rate-limiting precursor of carnosine, may also be PLP dependent. Thus, we examined the effects of dietary vitamin B6 on the muscle carnosine content of rats. Male and female rats were fed a diet containing 1, 7, or 35 mg pyridoxine (PN) HCl/kg for 6 weeks. Carnosine in skeletal muscles was quantified by ultra-performance liquid chromatography coupled with tandem mass spectrometry. In the gastrocnemius muscle of male rats, carnosine concentration was significantly higher in the 7 and 35 mg groups (+70 and +61%, respectively) than in the 1 mg PN HCl/kg group, whereas that in the soleus muscle of male rats was significantly higher only in the 7 mg group (+43%) than in the 1 mg PN HCl/kg group (P < 0.05). In both muscles of female rats, carnosine concentration was significantly higher in the 7 and 35 mg groups (+32 to +226%) than in the 1 mg PN HCl/kg group (P < 0.05). We also found that, compared to the 1 mg group, β-alanine concentrations in the 7 and 35 mg groups were markedly elevated in gastrocnemius muscles of male (+153 and +148%, respectively, P < 0.05) and female (+381 and +437%, respectively, P < 0.05) rats. Noteworthy, the concentrations of ornithine in the 7 and 35 mg groups were decreased in gastrocnemius muscles of male rats (−46 and −54%, respectively, P < 0.05), which strongly inversely correlated with β-alanine concentration (r = −0.84, P < 0.01). In humans, 19% lower muscle carnosine content was found in soleus muscle of women of the lower plasma PLP tertile, but this was not observed in gastrocnemius muscle or in men. We conclude that adequate dietary vitamin B6 is essential for maintaining carnosine in skeletal muscles of rats. Significantly lower soleus carnosine content among women close to PLP deficiency suggests that a similar phenomenon exists in the humans.

Coenzyme Q10 and Pyridoxal Phosphate Deficiency Is a Common Feature in Mucopolysaccharidosis Type III.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by deficiencies of lysosomal enzymes catalyzing degradation of glycosaminoglycans (GAGs). Previously, we reported a secondary plasma coenzyme Q10 (CoQ) deficiency in MPS patients. For this study, nine MPS patients were recruited in the Hospital Sant Joan de Déu (HSJD, Barcelona) and two patients in the Neurometabolic Unit, National Hospital (NMU, London), to explore the nutritional status of MPS type III patients by analyzing several vitamins and micronutrients in blood and in cerebrospinal fluid. Plasma CoQ and plasma and cerebrospinal fluid pyridoxal phosphate (PLP) content were analyzed by high-pressure liquid chromatography (HPLC) with electrochemical and fluorescence detection, respectively. We found that most MPS-III patients disclosed low plasma pyridoxal phosphate (PLP) values (seven out of nine) and also low plasma CoQ concentrations (eight out of nine). We observed significantly lower median values of PLP, tocopherol, and CoQ (Mann-Whitney U test, p = 0.006, p = 0.004, and p = 0.001, respectively) in MPS patients when compared with age-matched controls. Chi-square test showed a significant association between the fact of having low plasma PLP and CoQ values in the whole cohort of patients. Cerebrospinal fluid PLP values were clearly deficient in the two patients studied. In conclusion, we report a combined CoQ and PLP deficiency in MPS-III patients. These observations could be related to the complexity of the physiopathology of the disease. If our results are confirmed in larger series of patients, CoQ and PLP therapy could be trialed as coadjuvant therapy with the current MPS treatments.

Morton’s foot and pyridoxal 5′-phosphate deficiency: Genetically linked traits

Vitamin B6 is an essential vitamin needed for many chemical reactions in the human body. It exists as several vitamins forms but pyridoxal 5′-phosphate (PLP) is the phosphorylated form needed for transamination, deamination, and decarboxylation. PLP is important in the production of neurotransmitters, acts as a Schiff base and is essential in the metabolism of homocysteine, a toxic amino acid involved in cardiovascular disease, stroke, thrombotic and Alzheimer’s disease. This report announces the connection between a deficit of PLP with a genetically linked physical foot form known as the Morton’s foot. Morton’s foot has been associated with fibromyalgia/myofascial pain syndrome. Another gene mutation methylenetetrahydrofolate reductase (MTHFr) is now being recognized much commonly than previous with chronic fatigue, chronic Lyme diseases and as “the missing link” in other chronic diseases. PLP deficiency also plays a role in impaired glucose tolerance and may play a much bigger role in the obesity, diabetes, fatty liver and metabolic syndrome. Without the Schiff-base of PLP acting as an electron sink, storing electrons and dispensing them in the mitochondria, free radical damage occurs! The recognition that a phenotypical expression (Morton’s foot) of a gene resulting in deficiency of an important cofactor enzyme pyridoxal 5′-phosphate will hopefully alert physicians and nutritionist to these phenomena. Supplementation with PLP, L5-MTHF, B12 and trimethylglycine should be used in those patients with hyperhomocysteinemia and/or MTHFR gene mutation.

Sugar and Chromosome Stability: Clastogenic Effects of Sugars in Vitamin B6-Deficient Cells

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, has been implicated in preventing human pathologies, such as diabetes and cancer. However, the mechanisms underlying the beneficial effects of PLP are still unclear. Using Drosophila as a model system, we show that PLP deficiency, caused either by mutations in the pyridoxal kinase-coding gene (dPdxk) or by vitamin B6 antagonists, results in chromosome aberrations (CABs). The CAB frequency in PLP-depleted cells was strongly enhanced by sucrose, glucose or fructose treatments, and dPdxk mutant cells consistently displayed higher glucose contents than their wild type counterparts, an effect that is at least in part a consequence of an acquired insulin resistance. Together, our results indicate that a high intracellular level of glucose has a dramatic clastogenic effect if combined with PLP deficiency. This is likely due to an elevated level of Advanced Glycation End-products (AGE) formation. Treatment of dPdxk mutant cells with α-lipoic acid (ALA) lowered both AGE formation and CAB frequency, suggesting a possible AGE-CAB cause-effect relationship. The clastogenic effect of glucose in PLP-depleted cells is evolutionarily conserved. RNAi-mediated silencing of PDXK in human cells or treatments with PLP inhibitors resulted in chromosome breakage, which was potentiated by glucose and reduced by ALA. These results suggest that patients with concomitant hyperglycemia and vitamin B6 deficiency may suffer chromosome damage. This might impact cancer risk, as CABs are a well-known tumorigenic factor.

Vitamin B6 and the Immunity in Kidney Transplant Recipients

The study aimed to determine vitamin B6 status in elderly (age ≥ 60 years) and younger (age <60 years) recipients of allogeneic kidney graft and to investigate associations between vitamin B6 status and immunity markers. A retrospective observational study. The study was conducted at the Medical University of Gdańsk, Poland. We recruited 34 kidney allograft recipients (17 males and 17 females) and allocated them into 2 groups: patients aged ≥ 60 years (18 patients) and those aged <60 years (16 patients). Exclusion criteria included patients receiving vitamin B6 supplementation or drugs known to influence vitamin B6 metabolism. Plasma levels of pyridoxal 5'-phosphate (PLP), pyridoxal, pyridoxine, pyridoxamine, pyridoxamine 5'-phosphate, and 4 pyridoxic acid were determined by high-performance liquid chromatography. Measured immunity markers were serum cytokines (interleukin-6, interleukin-10, and transforming growth factor-β), levels of T-lymphocyte subsets, and the proliferative ability of peripheral blood mononuclear cells. Concentrations of all vitamin B6 vitamers in plasma (PLP, pyridoxal, pyridoxamine 5'-phosphate, pyridoxamine, pyridoxine, 4 pyridoxic acid) were comparable in the 2 studied groups. There were no cases of PLP deficiency in the study population, but 29% of patients had PLP concentrations more than the upper reference limit. Vitamin B6 vitamer concentrations were not influenced by gender, estimated glomerular filtration rate, and circulating phosphate concentration. There was no difference in immunity markers according to age. However, the plasma concentrations of vitamin B6 vitamers were inversely associated with levels of CD28(+) lymphocyte subsets, as well as with the proliferative response of peripheral blood mononuclear cells in both groups. No cases of vitamin B6 deficiency were found among kidney allograft recipients, and we report inverse links between vitamin B6 vitamer concentrations and markers of cellular immunity, suggesting that bioactive vitamin B6 concentration in kidney allograft recipients merits further investigation.

Neopterin, a Marker of Interferon-Gamma-Inducible Inflammation, Correlates with Pyridoxal-5′-Phosphate, Waist Circumference, HDL-Cholesterol, Insulin Resistance and Mortality Risk in Adult Boston Community Dwellers of Puerto Rican Origin

Interferon-gamma (IFNG), a pro-inflammatory cytokine, increases concentrations of neopterin, a stable pteridine derivative, due to IFNG-induced transcriptional activation of the rate-limiting enzyme of pteridines biosynthesis. Neopterin concentrations were reported to correlate with metabolic syndrome (MetS), the cause of increased mortality risk, in subjects of European ancestry. We were interested to assessed neopterin correlations with clinical markers of MetS and mortality risk in population with a different genetic background, i.e., Puerto Ricans residents of Boston. Since inflammation is associated with pyridoxal-5'-phosphate (PLP) deficiency, we assessed correlations of neopterin with PLP. Plasma neopterin concentrations were evaluated in 592 adult (45-75 years of age) participants of Boston Puerto Rican Health Study. Neopterin concentrations correlated with abdominal obesity (waist circumference, r = 0.085, p < 0.038), HDL cholesterol (r = -0.15, p < 0.0001), insulin resistance (HOMA-IR, r = 0.08, P < 0.03), and plasma pyridoxal-5'-phosphate (PLP (r = -0.13, P = 0.002). Neopterin concentrations of >16 nmol/L at baseline were associated with the increased risk of mortality in 113 subjects followed for 6 years. The present results together with previously reported data in European subjects suggest a similar pattern of neopterin correlations with MetS and mortality risk in population with different genetic backgrounds. PLP is a cofactor of IFNG-induced key enzymes of tryptophan-kynurenine metabolism. Since PLP deficiency is associated with the increased production of diabetogenic kynurenine derivative, xanthurenic acid, our results suggest that up-regulated IFNG production might contribute to the development of insulin resistance. Assessment of neopterin concentrations might help to monitor the activity of IFNG-inducible inflammation associated with aging-associated medical and psychiatric disorders.

Recent insights into pre‐ and postnatal pyridoxal phosphate deficiency, a treatable metabolic encephalopathy

Recent insights into pre‐ and postnatal pyridoxal phosphate deficiency, a treatable metabolic encephalopathy

Pyridoxal-5′-phosphate deficiency after intestinal and multivisceral transplantation

BackgroundSuccessful intestinal transplantation is measured by the achievement of clinical nutritional autonomy (CNA). However, the ability of the graft to maintain normal micronutrient levels including vitamins has yet to be thoroughly evaluated. ObjectiveAfter an initial clinical observation of isolated cases of pyridoxal-5′-phosphate (PLP) deficiency, this prospective study was designed to address the incidence of, risk factors for, and management of PLP deficiency in adult intestinal transplant recipients. DesignSerum PLP and homocysteine concentrations were prospectively measured before and after transplantation at frequent intervals. ResultsPLP deficiency occurred in 10% of candidates and in 96% of recipients within a median onset of 30 d (range: 4–118 d) after transplantation. Of this group, 41% were receiving parenteral nutrition (PN), 41% were receiving enteral feeding, and the remaining 18% had already achieved CNA. The overall cumulative risk was 24% at 15 d, 59% at 30 d, 79% at 45 d, and 90% at 90 d; none of the risk factors, including homocysteine concentrations, were significant. Nonetheless, the development of PLP deficiency during PN therapy was associated with a significant (P < 0.001) delay in the achievement of CNA. Despite development of severe deficiency in most cases, none of the subjects experienced clinical manifestations of PLP deficiency because of prompt replacement therapy. ConclusionsSerial monitoring of serum PLP concentrations is recommended for PN-dependent patients with short-bowel syndrome before and after transplantation for early detection and prompt initiation of preemptive therapy. Long-term measurement at frequent intervals is also recommended, particularly for transplant recipients, to diagnose late deficiency despite achievement of CNA and to prevent toxicity from overdose.

Predictive value of FIB-4 versus fibrotest, APRI, FIBROINDEX and FORNS to noninvasively estimate fibrosis in hepatitis C

Reply: We thank Dr. Adler's group for their interesting results which confirm the accuracy of the FIB-4 index for delineating significant (requiring therapy) and nonsignificant fibrosis in chronic hepatitis C (HCV), in HCV monoinfection as well as in human immunodeficiency virus (HIV)-HCV coinfection.1 Adler et al. included in their analysis a broad population of patients, some of them with chronic hepatitis B (HBV) infection and alcoholic liver disease. Our results in chronic hepatitis B (work submitted) suggest that the FIB-4 index is robust to diagnose nil-to-moderate fibrosis, providing therefore an interesting first-line evaluation. The comparison of the FIB-4 index with liver biopsies performed the same day in 138 patients allowed the correct identification of patients with nil-to-moderate fibrosis (METAVIR F0-F2) with an area under the receiving operating characteristic curve of 0.81 (confidence interval 95% 0.74-0.88) (P < 0.001). Interestingly, the longer the length of the liver biopsy, the higher was the AUROC increasing from 0.81 to 0.91. The FIB-4 score was less accurate for assessing the diagnosis of severe fibrosis (METAVIR F3-F4). The high number of African patients included in our study could explain at least in part these results: most discordant patients with a high FIB-4 index and nil-to-moderate fibrosis had a low platelet count, possibly following chronic malarial infection. In alcohol-related liver disease (ALD), our analysis of the FIB-4 index in 160 patients is disappointing and confirmed the idea that existing diagnostic tests based on aminotransferase level and platelet count are not suitable for the determination of fibrosis stage in patients with ALD (work submitted). This point could receive several nonexclusive explanations: alcohol is well known to induce a higher AST/ALT ratio than other causes of liver diseases; the level of aminotransferases is classically lower in ALD and this has been related to pyridoxal phosphate deficiency; platelet count is affected by the acute toxicity of ethanol on thrombopoiesis and its correlation to portal hypertension is therefore poor in that context. All these alcohol-related variations clearly make an impact on the FIB-4 index which includes AST, ALT, GGT and the platelet count. The accuracy and the low cost of the FIB-4 index should make the FIB-4 index, with the fibroscan, a first line noninvasive biochemical marker and should be added to the recommendations of the Haute Autorité de Santé.2 Anais Vallet-Pichard M.D.*, Vincent Mallet M.D., Ph.D.*, Stanislas Pol M.D., Ph.D.*, * Université Paris Descartes; INSERM U.567; APHP, Hôpital Cochin, Unité d'hépatologie Paris, France.

A Screen for Suppressors of Gross Chromosomal Rearrangements Identifies a Conserved Role for PLP in Preventing DNA Lesions

Genome instability is a hallmark of cancer cells. One class of genome aberrations prevalent in tumor cells is termed gross chromosomal rearrangements (GCRs). GCRs comprise chromosome translocations, amplifications, inversions, deletion of whole chromosome arms, and interstitial deletions. Here, we report the results of a genome-wide screen in Saccharomyces cerevisiae aimed at identifying novel suppressors of GCR formation. The most potent novel GCR suppressor identified is BUD16, the gene coding for yeast pyridoxal kinase (Pdxk), a key enzyme in the metabolism of pyridoxal 5′ phosphate (PLP), the biologically active form of vitamin B6. We show that Pdxk potently suppresses GCR events by curtailing the appearance of DNA lesions during the cell cycle. We also show that pharmacological inhibition of Pdxk in human cells leads to the production of DSBs and activation of the DNA damage checkpoint. Finally, our evidence suggests that PLP deficiency threatens genome integrity, most likely via its role in dTMP biosynthesis, as Pdxk-deficient cells accumulate uracil in their nuclear DNA and are sensitive to inhibition of ribonucleotide reductase. Since Pdxk links diet to genome stability, our work supports the hypothesis that dietary micronutrients reduce cancer risk by curtailing the accumulation of DNA damage and suggests that micronutrient depletion could be part of a defense mechanism against hyperproliferation.

Pyridoxine-responsive seizures as the first symptom of infantile hypophosphatasia caused by two novel missense mutations (c.677T > C, p.M226T; c.1112C > T, p.T371I) of the tissue-nonspecific alkaline phosphatase gene

Pyridoxine-responsive seizures (PRS) and the role of pyridoxine (PN, vitamin B 6) in hypophosphatasia (HPP) are incompletely understood. Typically, PRS and HPP are rare, independent, metabolic disorders. In PRS, seizures resist standard anticonvulsants apart from PN, yet have a good prognosis. In HPP, inactivation of the tissue nonspecific isoenzyme of alkaline phosphatase (TNSALP) impairs skeletal mineralization and causes rickets in infants that can be fatal. Here, we report a 7-month-old girl, newly diagnosed with infantile HPP, who presented as a neonate with PRS but without bony abnormalities. Analysis of biogenic amines in cerebrospinal fluid (CSF) suggested brain pyridoxal 5′-phosphate (PLP) deficiency, although PLP in CSF was not decreased. She had normal cognitive milestones but failure to thrive and rickets. Nearly undetectable serum ALP activity, elevated plasma PLP and urinary phosphoethanolamine (PEA) and inorganic pyrophosphate (PPi) levels, hypercalcemia, hypercalciuria and nephrocalcinosis were consistent with infantile HPP. Only prednisolone reduced serum calcium levels. Despite improved growth and weight gain, she developed rib fractures and died from respiratory failure at age 9 months. Sequence analysis of the TNSALP gene revealed novel missense mutations in exon 7 (c.677T > C, p.M226T) and exon 10 (c.1112C > T, p.T371I). Our patient demonstrated that PRS in neonates may not necessarily be “idiopathic”; instead, such seizures can be caused by severe HPP that becomes clinically apparent later in infancy. The pathophysiology of PRS in HPP differs from the three other genetic defects known to cause PRS, but all may lead to brain PLP deficiency reducing seizure thresholds. All reported HPP patients with neonatal seizures died within 18 months of birth, suggesting that PRS is an indicator of HPP severity and lethal prognosis. We recommend that assessment of any neonate with PRS should include measurement of serum ALP activity.

Inborn errors affecting vitamin B 6 metabolism

Vitamin B6is an important vitamin for normal brain function. The metabolism of dietary vitamin B6to its active cofactor pyridoxal 5´-phosphate is described. The mechanism of action of pyridoxal 5´-phosphate is described, as are some important functions in the brain. The clinical features and biochemistry of three inborn errors of metabolism affecting brain pyridoxal 5´-phosphate concentrations are described, each of which cause early-onset epilepsy of variable severity. These are pyridoxine phosphate oxidase deficiency, hyperprolinemia Type 2 and pyridoxine-dependent epilepsy caused by antiquitin deficiency. Hypophosphatasia is also discussed briefly, as the epilepsy that can complicate this disorder appears to be due to pyridoxal phosphate deficiency. Lastly, the antiepileptic properties of pyridoxine and pyridoxal phosphate are discussed.

Biochemical and molecular characterization of 18 patients with pyridoxine-dependent epilepsy and mutations of the antiquitin (ALDH7A1) gene

Patients with pyridoxine dependent epilepsy (PDE) present with early-onset seizures resistant to common anticonvulsants. According to the benefit of pyridoxine (vitamin B(6)) and recurrence of seizures on pyridoxine withdrawal, patients so far have been classified as having definite, probable, or possible PDE. Recently, PDE has been shown to be caused by a defect of alpha-amino adipic semialdehyde (AASA) dehydrogenase (antiquitin) in the cerebral lysine degradation pathway. The accumulating compound piperideine-6-carboxylic acid (P6C) was shown to inactivate pyridoxalphosphate (PLP) by a Knoevenagel condensation. Pipecolic acid (PA) and AASA are markedly elevated in urine, plasma, and cerebrospinal fluid (CSF) and thus can be used as biomarkers of the disease. We have investigated 18 patients with neonatal seizure onset, who have been classified as having definite (11), probable (four), or possible (three) PDE. All patients had elevated PA and AASA in plasma (and urine) while on treatment with individual dosages of pyridoxine. Within this cohort, molecular analysis identified 10 novel mutations (six missense mutations, one nonsense mutation, two splice site mutations) within highly conserved regions of the antiquitin gene. Seven mutations were located in exonic sequences and two in introns 7 and 17. Furthermore, a novel deletion of exon 7 was identified. Two of the 36 alleles investigated require further investigation. A known mutation (p.Glu399Gln) was found with marked prevalence, accounting for 12 out of 36 alleles (33%) within our cohort. Pyridoxine withdrawal is no longer needed to establish the diagnosis of "definite" PDE. Administration of pyridoxine in PDE may not only correct secondary PLP deficiency, but may also lead to a reduction of AASA (and P6C) as presumably toxic compounds.