Multiomic analysis in fibroblasts of patients with inborn errors of cobalamin metabolism reveals concordance with clinical and metabolic variability.

BACKGROUND: The high variability in clinical and metabolic presentations of inborn errors of cobalamin (cbl) metabolism (IECM), such as the cblC/epicblC types with combined deficits in methylmalonyl-coA mutase (MUT) and methionine synthase (MS), are not well understood. They could be explained by the impaired expression/activity of enzymes from other metabolic pathways. METHODS: We performed metabolomic, genomic, proteomic, and post-translational modification (PTM) analyses in fibroblasts from three cblC cases and one epi-cblC case compared with three cblG cases with specific MS deficits and control fibroblasts. FINDINGS: CblC patients had metabolic profilings consistent with altered urea cycle, glycine, and energy mitochondrial metabolism. Metabolomic analysis showed partial disruption and increased glutamate/ketoglutarate anaplerotic pathway of the tricarboxylic acid cycle (TCA), in patient fibroblasts. RNA-seq analysis showed decreased expression of MT-TT (mitochondrial tRNA threonine), MT-TP (mitochondrial tRNA proline), OXCT1 (succinyl CoA:3-oxoacid CoA transferase deficiency), and MT-CO1 (cytochrome C oxidase subunit 1). Proteomic changes were observed for key mitochondrial enzymes, including NADH:ubiquinone oxidoreductase subunit A8 (NDUFA8), carnitine palmitoyltransferase 2 (CPT2), and ubiquinol-cytochrome C reductase, complex III subunit X (UQCR10). Propionaldehyde addition in ornithine aminotransferase was the predominant PTM in cblC cells and could be related with the dramatic cellular increase in propionate and methylglyoxalate. It is consistent with the decreased concentration of ornithine reported in 3 cblC cases. Whether the changes detected after multi-omic analyses underlies clinical features in cblC and cblG types of IECM, such as peripheral and central neuropathy, cardiomyopathy, pulmonary hypertension, development delay, remains to be investigated. INTERPRETATION: The omics-related effects of IECM on other enzymes and metabolic pathways are consistent with the diversity and variability of their age-related metabolic and clinical manifestations. PTMs are expected to produce cumulative effects, which could explain the influence of age on neurological manifestations. FUNDING: French Agence Nationale de la Recherche (Projects PREDICTS and EpiGONE) and Inserm.

Blood phenylalanine fluctuation in phenylketonuric children treated by BH4 or low-phenylalanine diet from birth.

The prognosis of phenylketonuria (PKU) is related to the quality of metabolic control all life-long. PKU treatment is based on a low-Phe diet, 6R-tetrahydrobiopterin (BH4) treatment for the BH4-responsive PKU patients or enzyme replacement therapy. Fluctuations in blood phenylalanine (Phe) concentrations may be an important determinant of intellectual outcome in patients with early and continuously treated phenylketonuria (PKU). The aim of this work is to study the fluctuation of Blood Phe in patients treated by BH4 from birth in comparison with patients treated by low-Phe diet. We conducted a retrospective study in a national reference center for PKU management. We compared mean phenylalanine blood concentration and its fluctuation in 10 BH4-responder patients (BH4R) and in 10 BH4 non-responder patients (BH4NR) treated from birth. The mean blood Phe concentration is similar between the two groups before 10 years of age (290 ± 135 (BH4R) vs. 329 ± 187 µmol/L, p = 0.066 (BH4NR)) while it is lower in the BH4R group after 10 years of age. (209 ± 69 vs. 579 ± 136 µmol/L, p = 0.0008). Blood Phe fluctuation is significantly lower in the BH4R group compared to the BH4NR group (70.2 ± 75.6 vs. 104.4 ± 111.6 µmol/L, p < 0.01) before 6 years of age. There are no significant differences observed on nutritional status, growth, and neuropsychological tests between the two groups. BH4 introduced in the neonatal period is associated with less blood Phe fluctuation before 6 years. Additional time and patients are required to determine if the decrease in Phe fluctuation would positively impact the long-term outcome of PKU patients.

[POCT-management during the first wave of Covid-19 in France. Results of a national survey leaded by the SFBC-POCT Working Group]. / Gestion de la biologie délocalisée lors de la première vague de Covid-19 en France. Résultats de l'enquête nationale menée par le groupe de travail des EBMD de la SFBC.

During the first wave of Covid-19 in France, in spring 2020, healthcare institution's laboratory had to adapt itself quickly to the growing demand for emergency biology, in particular by reorganizing their POCT analyzers: redeployment of analyzers and/or new installations. In order to analyze this management, a subgroup of 15 hospital biologists from the SFBC Working Group "Biochemical markers of Covid-19" sent, in fall 2020, an on-line survey to French hospital laboratories using POCT. Answers analysis (n = 86) shows a territorial disparity related to the severity of the first wave: increased activity essentially in red zones, management of unexpected situations, training of additional nursing staff for 40 % of the laboratories... The survey also showed simplification of aspects related to accreditation those periods of health crisis. An additional survey, carried out in the spring of 2021, showed good overall satisfaction of the healthcare services (n = 139) concerning the services provided by biology in the POCT sector. Because of their great adaptation capacity, the laboratories and their POCT-teams have played a key role in the management of the first wave of Covid-19 in France. However, the success of these organizations requires an essential collaboration between laboratories and healthcare services. The results of this survey are fundamental in the context of the prolongation of the pandemia throughout the world with a POCT sector appearing to be growing.

[Newborn screening of phenylketonuria in France]. / Le dépistage de la phénylcétonurie en France.

Phenylketonuria is the most common inborn error of metabolism and causes irreversible mental retardation if left untreated. Its newborn screening was made possible by the technique of blood collection on filter paper developed by Robert Guthrie. Neonatal PKU screening began in France in the early 1970s. It was initially carried out by a bacteriological method, then by fluorometry, and finally, since 2020, by tandem mass spectrometry. More than 35 million newborns have been screened to date. This resulted in the diagnosis of more than 3,500 children with PKU or mild hyperphenylalaninemia. The management of these children has improved over time, in particular thanks to the techniques of biochemistry and molecular genetics which lead to an accurate diagnosis and the arrival of drug treatment by sapropterin. Thanks to this screening, which allows for early management, the prognosis of PKU has been transformed and, although neurological or behavioral problems may arise, these patients are living normally today.

TITLE: Le dépistage de la phénylcétonurie en France. ABSTRACT: La phénylcétonurie (PCU) est la plus fréquente des erreurs innées du métabolisme et entraîne un retard mental irréversible en l'absence de traitement. Son dépistage néonatal a été rendu possible grâce à la technique de recueil de sang sur papier buvard mise au point par Robert Guthrie. Le dépistage néonatal de la PCU a débuté en France au début des années 1970. Il a été initialement réalisé par une technique bactériologique, puis fluorimétrique et, enfin, depuis 2020 par spectrométrie de masse en tandem. Plus de 35 millions de nouveau-nés ont été dépistés à ce jour, ce qui a permis de diagnostiquer plus de 3 500 enfants porteurs de PCU ou hyperphénylalaninémie modérée. La prise en charge de ces enfants a évolué avec le temps, en particulier grâce aux techniques de biochimie et de génétique moléculaire qui permettent un diagnostic précis et grâce à l'arrivée d'un traitement médicamenteux par saproptérine. Grâce à ce dépistage, qui permet une prise en charge précoce, le pronostic de la PCU a été transformé et, même s'il peut survenir des problèmes neurologiques ou comportementaux, ces patients ont une vie normale aujourd'hui.

Cardiovascular manifestations of intermediate and major hyperhomocysteinemia due to vitamin B12 and folate deficiency and/or inherited disorders of one-carbon metabolism: a 3.5-year retrospective cross-sectional study of consecutive patients.

BACKGROUND: The association of moderate hyperhomocysteinemia (HHcy) (15-30 µmol/L) with cardiovascular diseases (CVD) has been challenged by the lack of benefit of vitamin supplementation to lowering homocysteine. Consequently, the results of interventional studies have confused the debate regarding the management of patients with intermediate/severe HHcy. OBJECTIVE: We sought to evaluate the association of intermediate (30-100 µmol/L) and severe (>100 µmol/L) HHcy related to vitamin deficiencies and/or inherited disorders with CVD outcomes. METHODS: We performed a retrospective cross-sectional study on consecutive patients who underwent a homocysteine assay in a French University Regional Hospital Center. Patients with CVD outcomes were assessed for vitamin B12, folate, Hcy, methylmalonic acid, and next-generation clinical exome sequencing. RESULTS: We evaluated 165 patients hospitalized for thromboembolic and other cardiovascular (CV) manifestations among 1006 patients consecutively recruited. Among them, 84% (138/165) had Hcy >30 µmol/L, 27% Hcy >50 µmol/L (44/165) and 3% Hcy >100 µmol/L (5/165). HHcy was related to vitamin B12 and/or folate deficiency in 55% (87/165), mutations in one or more genes of one-carbon and/or vitamin B12 metabolisms in 11% (19/165), and severe renal failure in 15% (21/141) of the studied patients. HHcy was the single vascular risk retrieved in almost 9% (15/165) of patients. Sixty % (101/165) of patients received a supplementation to treat HHcy, with a significant decrease in median Hcy from 41 to 17 µmol/L (IQR: 33.6-60.4 compared with 12.1-28). No recurrence of thromboembolic manifestations was observed after supplementation and antithrombotic treatment of patients who had HHcy as a single risk, after ∼4 y of follow-up. CONCLUSION: The high frequency of intermediate/severe HHcy differs from the frequent moderate HHcy reported in previous observational studies of patients with pre-existing CVD. Our study points out the importance of diagnosing and treating nutritional deficiencies and inherited disorders to reverse intermediate/severe HHcy associated with CVD outcomes.

The spectrum of biochemical alterations associated with organ dysfunction and inflammatory status and their association with disease outcomes in severe COVID-19: A longitudinal cohort and time-series design study.

BACKGROUND: In patients with severe COVID-19, no data are available on the longitudinal evolution of biochemical abnormalities and their ability to predict disease outcomes. METHODS: Using a retrospective, longitudinal cohort study design on consecutive patients with severe COVID-19, we used an extensive biochemical dataset of serial data and time-series design to estimate the occurrence of organ dysfunction and the severity of the inflammatory reaction and their association with acute respiratory failure (ARF) and death. FINDINGS: On the 162 studied patients, 1151 biochemical explorations were carried out for up to 59 biochemical markers, totaling 15,260 biochemical values. The spectrum of biochemical abnormalities and their kinetics were consistent with a multi-organ involvement, including lung, kidney, heart, liver, muscle, and pancreas, along with a severe inflammatory syndrome. The proportion of patients who developed an acute kidney injury (AKI) stage 3, increased significantly during follow-up (0·9%, day 0; 21·4%, day 14; P<0·001). On the 20 more representative biochemical markers (>250 iterations), only CRP >90 mg/L (odds ratio [OR] 6·87, 95% CI, 2·36-20·01) and urea nitrogen >0·36 g/L (OR 3·91, 95% CI, 1·15-13·29) were independently associated with the risk of ARF. Urea nitrogen >0·42 g/L was the only marker associated with the risk of COVID-19 related death. INTERPRETATION: Our results point out the lack of the association between the inflammatory markers and the risk of death but rather highlight a significant association between renal dysfunction and the risk of COVID-19 related acute respiratory failure and death.

[Phenylketonuria, from diet to gene therapy]. / La phénylcétonurie - De la diététique à la thérapie génique.

The prognosis for phenylketonuria (PKU) has been improved by neonatal screening and dietary management via a low-phenylalanine diet. This treatment must be followed throughout life, which induces severe compliance problems. Drug treatment with sapropterin (or BH4) has come to help a reduced percentage of patients who respond to this drug. A subcutaneous enzyme therapy is available in the USA and has obtained European marketing authorization, but generates significant side effects, which limits its effectiveness. New therapeutic options for PKU are currently being developed, in particular gene therapy. The purpose of this article is to take stock of the pathophysiology and the various new therapeutic modalities currently in development.

Title: La phénylcétonurie - De la diététique à la thérapie génique. Abstract: Le pronostic de la phénylcétonurie (PCU) a été transformé par le dépistage néonatal et la prise en charge diététique via un apport contrôlé en phénylalanine. Ce traitement doit être suivi toute la vie durant, ce qui pose des problèmes de compliances importants. Un traitement médicamenteux par saproptérine (ou BH4) est venu apporter une aide à un pourcentage réduit de patients qui répondent à ce médicament. Une enzymothérapie par voie sous-cutanée est disponible aux États-Unis et a obtenue une AMM européenne, mais génère des effets secondaires importants, ce qui en limite l'efficacité. De nouvelles options thérapeutiques de la PCU sont actuellement en développement, en particulier par thérapie génique. Le but de cet article est de faire le point sur la physiopathologie et sur les différentes nouvelles modalités thérapeutiques actuellement en développement.

Long-term ACE Inhibitor/ARB Use Is Associated With Severe Renal Dysfunction and Acute Kidney Injury in Patients With Severe COVID-19: Results From a Referral Center Cohort in the Northeast of France.

BACKGROUND: In patients with severe coronavirus disease 2019 (COVID-19), data are scarce and conflicting regarding whether chronic use of angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) influences disease outcomes. In patients with severe COVID-19, we assessed the association between chronic ACEI/ARB use and the occurrence of kidney, lung, heart, and liver dysfunctions and the severity of the inflammatory reaction as evaluated by biomarkers kinetics, and their association with disease outcomes. METHODS: We performed a retrospective longitudinal cohort study on consecutive patients with newly diagnosed severe COVID-19. Independent predictors were assessed through receiver operating characteristic analysis, time-series analysis, logistic regression analysis, and multilevel modeling for repeated measures. RESULTS: On the 149 patients included in the study 30% (44/149) were treated with ACEI/ARB. ACEI/ARB use was independently associated with the following biochemical variations: phosphorus >40 mg/L (odds ratio [OR], 3.35, 95% confidence interval [CI], 1.83-6.14), creatinine >10.1 mg/L (OR, 3.22, 2.28-4.54), and urea nitrogen (UN) >0.52 g/L (OR, 2.65, 95% CI, 1.89-3.73). ACEI/ARB use was independently associated with acute kidney injury stage ≥1 (OR, 3.28, 95% CI, 2.17-4.94). The daily dose of ACEI/ARB was independently associated with altered kidney markers with an increased risk of +25 to +31% per each 10 mg increment of lisinopril-dose equivalent. In multivariable multilevel modeling, UN >0.52 g/L was independently associated with the risk of acute respiratory failure (OR, 3.54, 95% CI, 1.05-11.96). CONCLUSIONS: Patients chronically treated with ACEI/ARB who have severe COVID-19 are at increased risk of acute kidney injury. In these patients, the increase in UN associated with ACEI/ARB use could predict the development of acute respiratory failure.

Efficacy of low dose nitisinone in the management of alkaptonuria.

AIM: To study the efficacy of low dosage of nitisinone in alkaptonuria. BACKGROUND: Alkaptonuria (AKU) is a rare genetic disease which induces deposition of homogentisic acid (HGA) in connective inducing premature arthritis, lithiasis, cardiac valve disease, fractures, muscle and tendon ruptures and osteopenia. Recent studies showed that nitisinone decreases HGA and is a beneficial therapy in AKU. This treatment induces an increase in tyrosine levels which can induces adverse effects as keratopathy. METHODS: We described the evolution HGA excretion and tyrosine evolution in 3 AKU patients treated by very low dosage of nitisinone with regards to their daily protein intakes. We also described the first pregnancy in an AKU patient treated by nitisinone. RESULTS: We found mild clinical signs of alkaptonuria on vertebra MRI in two young adults and homogentisate deposition in teeth of a 5 years old girl. Very low dose of nitisinone (10% of present recommended dose: 0.2 mg/day) allowed to decrease homogentisic acid by >90% without increasing tyrosine levels above 500 µmol/ in these three patients. INTERPRETATIONS: The analysis of the follow-up data shows that, in our three patients, a low-dosage of nitisinone is sufficient to decrease urinary HGA without increasing plasma tyrosine levels above the threshold of 500 µmol/L.

Cystathionine ß-synthase genetic variant rs2124459 is associated with a reduced risk of cleft palate in French and Belgian populations.

BACKGROUND: Orofacial cleft (OFC) is the most prevalent craniofacial birth defect. Genes involved in one-carbon, folate and vitamin B12 metabolisms have been associated with OFC but no study performed a concomitant assessment on genes involved in these three pathways. OBJECTIVE: We looked for potential genetic variants associated with OFC using an exhaustive gene panel of one-carbon metabolism. METHODS: We performed a case-control discovery study on children with OFC (236 cases, 145 controls) and their related mothers (186 cases, 127 controls). We performed a replication study on the top significant genetic variant in an independent group from Belgium (248 cases, 225 controls). RESULTS: In the discovery study on 'mothers', the CBS locus reached array-wide significance (p=9.13×10-6; Bonferroni p=4.77×10-3; OR 0.47 (0.33 to 0.66)) among the 519 haplotypes tested for their association with OFC risk. Within the CBS haplotype block (rs2124459, rs6586282, rs4920037, rs234705, rs234709), the rs2124459 was the most significantly associated with a reduced risk of OFC (p=1.77×10-4; Bonferroni p=2.00×10-2; OR 0.53 (0.38 to 0.74), minor allele). The rs2124459 was associated with a reduced risk of cleft palate (CP) (p=6.78×10-5; Bonferroni p=7.80×10-3; OR 0.40 (0.25 to 0.63)). In the 'children' group, the rs2124459 was associated with a reduced risk of CP (p=0.02; OR 0.61 (0.40 to 0.93), minor allele). The association between rs2124459 and reduced risk of CP was replicated in an independent children population from Belgium (p=0.02; OR 0.64 (0.44 to 0.93), minor allele). CONCLUSIONS: The CBS rs2124459 was associated with a reduced risk of CP in both French and Belgian populations. These results highlight the prominent involvement of the vitamin B6-dependent transsulfuration pathway of homocysteine in OFC risk and the interest for evaluating vitamin B6 status in further population studies.

Folate- and vitamin B12-deficient diet during gestation and lactation alters cerebellar synapsin expression via impaired influence of estrogen nuclear receptor α.

Deficiency in the methyl donors vitamin B12 and folate during pregnancy and postnatal life impairs proper brain development. We studied the consequences of this combined deficiency on cerebellum plasticity in offspring from rat mothers subjected to deficient diet during gestation and lactation and in rat neuroprogenitor cells expressing cerebellum markers. The major proteomic change in cerebellum of 21-d-old deprived females was a 2.2-fold lower expression of synapsins, which was confirmed in neuroprogenitors cultivated in the deficient condition. A pathway analysis suggested that these proteomic changes were related to estrogen receptor α (ER-α)/Src tyrosine kinase. The influence of impaired ER-α pathway was confirmed by abnormal negative geotaxis test at d 19-20 and decreased phsophorylation of synapsins in deprived females treated by ER-α antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP). This effect was consistent with 2-fold decreased expression and methylation of ER-α and subsequent decreased ER-α/PPAR-γ coactivator 1 α (PGC-1α) interaction in deficiency condition. The impaired ER-α pathway led to decreased expression of synapsins through 2-fold decreased EGR-1/Zif-268 transcription factor and to 1.7-fold reduced Src-dependent phosphorylation of synapsins. The treatment of neuroprogenitors with either MPP or PP1 (4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline, 6,7-dimethoxy-N-(4-phenoxyphenyl)-4-quinazolinamine, SKI-1, Src-l1) Src inhibitor produced similar effects. In conclusion, the deficiency during pregnancy and lactation impairs the expression of synapsins through a deregulation of ER-α pathway.

Early methyl donor deficiency alters cAMP signaling pathway and neurosteroidogenesis in the cerebellum of female rat pups.

Early deficiency of the methyl donors folate and vitamin B12 produces hyperhomocysteinemia and cognitive and motor disorders in 21-day-old rat pups from dams fed a diet deficient in methyl donors during gestation and lactation. These disorders are associated with impaired neurogenesis and altered synaptic plasticity in cerebellum. We aimed to investigate whether these disorders could be related to impaired expression of neurosteroidogenesis-associated proteins, key regulator receptors, and some steroid content in the cerebellum. The methyl donor deficiency produced a decreased concentration of folate and vitamin B12, along with accumulation of homocysteine in Purkinje cells in both sexes, whereas the S-adenosylmethionine/S-adenosylhomocysteine ratio was reduced only in females. The transcription level and protein expression of StAR, aromatase, ERα, ERß, and LH receptors were decreased only in females, with a marked effect in Purkinje cells, as shown by immunohistochemistry. Consistently, reduced levels of estradiol and pregnenolone were measured in cerebellar extracts of females only. The decreased expression levels of the transcriptional factors CREB, phospho-CREB, and SF-1, the lesser increase of cAMP concentration, and the lower level of phospho-PKC in the cerebellum of deficient females suggest that the activation of neurosteroidogenesis via cAMP-mediated signaling pathways associated with LHR activation would be altered. In conclusion, a gestational methyl donor deficiency impairs neurosteroidogenesis in cerebellum in a sex-dependent manner.

Gastric intrinsic factor deficiency with combined GIF heterozygous mutations and FUT2 secretor variant.

Several genome-wide association studies (GWAS) have identified a strong association between serum vitamin B12 and fucosyltransferase 2 (FUT2), a gene associated with susceptibility to Helicobacter pylori infection. Hazra et al. conducted a meta-analysis of three GWAS and found three additional loci in MUT, CUBN and TCN1. Other GWAS conducted in Italy and China confirmed the association for FUT2 gene. Alpha-2-fucosyltransferase (FUT2) catalyzes fucose addition to form H-type antigens in exocrine secretions. FUT2 non-secretor variant produces no secretion of H-type antigens and is associated with high-plasma vitamin B12 levels. This association was explained by the influence of FUT2 on H. pylori, which is a risk factor of gastritis, a main cause of vitamin B12 impaired absorption. However, we recently showed that H. pylori serology had no influence on FUT2 association with vitamin B12, in a large sample population, suggesting the involvement of an alternative mechanism. GIF is another gene associated with plasma levels of vitamin B12 and gastric intrinsic factor (GIF) is a fucosylated protein needed for B12 absorption. Inherited GIF deficiency produces B12 deficiency unrelated with gastritis. We report 2 families with heterozygous GIF mutation, 290T>C, M97T, with decreased binding affinity of GIF for vitamin B12 and one family with heterozygous GIF mutation 435_437delGAA, K145_N146delinsN and no B12 binding activity of mutated GIF. All cases with vitamin B12 deficit carried the FUT2 rs601338 secretor variant. Ulex europeus binding to GIF was influenced by FUT2 genotypes and GIF concentration was lower, in gastric juice from control subjects with the secretor genotype. GIF290C allele was reported in 5 European cases and no Africans among 1282 ambulatory subjects and was associated with low plasma vitamin B12 and anaemia in the single case bearing the FUT2 secretor variant. We concluded that FUT2 secretor variant worsens B12 status in cases with heterozygous GIF mutations by impairing GIF secretion, independently from H. pylori-related gastritis.

Early methyl donor deficiency produces severe gastritis in mothers and offspring through N-homocysteinylation of cytoskeleton proteins, cellular stress, and inflammation.

We examined the gastric mucosa structure and inflammatory status in control well-nourished Wistar dams and in Wistar dams deprived of choline, folate, and vitamin B12 during gestation and suckling periods, and in their offspring just before birth and at weaning. In this model of methyl donor deficiency (MDD), structural protein (E-cadherin and actin) N-homocysteinylation was measured through immunoprecipitation and proximity ligation assays. Cellular stress, inflammation, and apoptosis were estimated by the analysis of the NF-κB pathway, and the expression of superoxide dismutase, cyclooxygenase-2, tumor necrosis factor α, caspases 3 and 9, and TUNEL assay. Aberrant gastric mucosa formation and signs of surface layer erosion were detected in MDD fetuses and weanlings. E-cadherin and actin were N-homocysteinylated (+215 and +249% vs. controls, respectively; P<0.001). Expression of ß-catenin staining drastically decreased (-98%; P<0.01). NF-κB pathway was activated (+124%; P<0.01). Expressions of all inflammatory factors (+70%; P<0.01), superoxide dismutase (+55%; P<0.01), and caspases (+104%; P<0.01) were markedly increased. These changes were also observed in dams, to a lesser extent. Early MDD induced gastric mucosa injury similar to atrophic gastritis through structural protein N-homocysteinylation, marked inflammation, and apoptosis, despite activation of repair machinery.

Increased homocysteinemia is associated with beneficial effects on body weight after long-term high-protein, low-fat diet in rats.

OBJECTIVE: Diets rich in protein are often used for weight loss in obese patients, but their long-term effects are not fully understood. Homocysteine (Hcy) is considered to be a risk factor for cardiovascular diseases, and its levels are influenced by diet, particularly the protein and fat content. We studied the effect of diets with varying fat/protein content on body weight and composition, food intake, Hcy, B vitamins, leptin, and several pro-inflammatory cytokines. METHODS: For 2 mo, Long-Evans rats were fed either a low protein/high fat (LP), a standard control (C), or a high protein/low fat (HP) diet containing 5, 15, or 40% protein, respectively, and normal carbohydrate content (55% of total energy). RESULTS: The HP rats ingested 12 to 15% fewer calories (P < 0.001), gained less weight (P < 0.04), and were less fatty (P < 0.01) than the other groups. Plasma Hcy was increased in HP rats compared to C (+23%) and LP (+29%) rats (P < 0.03). It was positively correlated with protein intake (r = 0.386; P < 0.01). No obvious signs of inflammation were observed in any of the groups. Hcy increase was related directly to decrease in plasma folate (r = -0.372; P < 0.02). CONCLUSION: These data confirm the beneficial effects of HP diets on body weight but bring attention to the control of folate allowance to limit the adverse effects of elevated Hcy. Ingestion of folate-rich foods or even folate supplementation should be considered when using these HP diets over the long term for weight loss.

Methyl donor deficiency impairs fatty acid oxidation through PGC-1α hypomethylation and decreased ER-α, ERR-α, and HNF-4α in the rat liver.

BACKGROUND & AIMS: Folate and cobalamin are methyl donors needed for the synthesis of methionine, which is the precursor of S-adenosylmethionine, the substrate of methylation in epigenetic, and epigenomic pathways. Methyl donor deficiency produces liver steatosis and predisposes to metabolic syndrome. Whether impaired fatty acid oxidation contributes to this steatosis remains unknown. METHODS: We evaluated the consequences of methyl donor deficient diet in liver of pups from dams subjected to deficiency during gestation and lactation. RESULTS: The deprived rats had microvesicular steatosis, with increased triglycerides, decreased methionine synthase activity, S-adenosylmethionine, and S-adenosylmethionine/S-adenosylhomocysteine ratio. We observed no change in apoptosis markers, oxidant and reticulum stresses, and carnityl-palmitoyl transferase 1 activity, and a decreased expression of SREBP-1c. Impaired beta-oxidation of fatty acids and carnitine deficit were the predominant changes, with decreased free and total carnitines, increased C14:1/C16 acylcarnitine ratio, decrease of oxidation rate of palmitoyl-CoA and palmitoyl-L-carnitine and decrease of expression of novel organic cation transporter 1, acylCoA-dehydrogenase and trifunctional enzyme subunit alpha and decreased activity of complexes I and II. These changes were related to lower protein expression of ER-α, ERR-α and HNF-4α, and hypomethylation of PGC-1α co-activator that reduced its binding with PPAR-α, ERR-α, and HNF-4α. CONCLUSIONS: The liver steatosis resulted predominantly from hypomethylation of PGC1-α, decreased binding with its partners and subsequent impaired mitochondrial fatty acid oxidation. This link between methyl donor deficiency and epigenomic deregulations of energy metabolism opens new insights into the pathogenesis of fatty liver disease, in particular, in relation to the fetal programming hypothesis.

Helicobacter pylori serologic status has no influence on the association between fucosyltransferase 2 polymorphism (FUT2 461 G->A) and vitamin B-12 in Europe and West Africa.

BACKGROUND: Genomewide association studies have shown a relation between plasma vitamin B-12 concentration and the 461G→A polymorphism of fucosyltransferase 2 (FUT2), a gene associated with susceptibility to Helicobacter pylori infection. OBJECTIVE: We evaluated in 2 populations the association of FUT2 461 G→A polymorphism with vitamin B-12 and related metabolic markers and investigated whether the influence of FUT2 on H. pylori serology is part of the mechanisms that underlie these associations. DESIGN: The study included 1282 ambulatory subjects from Europe and West Africa. Blood concentrations of vitamin B-12, folate, homocysteine, and methylmalonic acid were measured. Genotyping was performed by real-time polymerase chain reaction. H. pylori serology testing was performed by using ELISA. RESULTS: In univariate analysis, FUT2 461 A/A genotype was associated with higher plasma vitamin B-12 concentration in the total population (P = 0.0007) as well as in Europe (P = 0.0009) and in West Africa (P = 0.0015). Positivity for H. pylori serology was higher in West Africa (P < 0.0001) and was not associated with low plasma vitamin B-12. The prevalence of H. pylori-positive patients did not differ among FUT2 461 G→A genotypes (P = 0.2068). In multivariate analysis, FUT2 461 G→A genotype (P = 0.0008), but not positive H. pylori serology, was an independent predictor of plasma vitamin B-12 concentration. CONCLUSION: This study confirms the influence of FUT2 461 G→A polymorphism on plasma vitamin B-12 concentration and showed no influence of H. pylori serologic status on this association in ambulatory subjects from Europe and West Africa.

Methyl donor deficiency induces cardiomyopathy through altered methylation/acetylation of PGC-1α by PRMT1 and SIRT1.

Cardiomyopathies occur by mechanisms that involve inherited and acquired metabolic disorders. Both folate and vitamin B12 deficiencies are associated with left ventricular dysfunction, but mechanisms that underlie these associations are not known. However, folate and vitamin B12 are methyl donors needed for the synthesis of S-adenosylmethionine, the substrate required for the activation by methylation of regulators of energy metabolism. We investigated the consequences of a diet lacking methyl donors in the myocardium of weaning rats from dams subjected to deficiency during gestation and lactation. Positron emission tomography (PET), microscope and metabolic examinations evidenced a myocardium hypertrophy, with cardiomyocyte enlargement, disturbed mitochondrial alignment, lipid droplets, decreased respiratory activity of complexes I and II and decreased S-adenosylmethionine:S-adenosylhomocysteine ratio. The increased concentrations of triglycerides and acylcarnitines were consistent with a deficit in fatty acid oxidation. These changes were explained by imbalanced acetylation/methylation of PGC-1α, through decreased expression of SIRT1 and PRMT1 and decreased S-adenosylmethionine:S-adenosylhomocysteine ratio, and by decreased expression of PPARα and ERRα. The main changes of the myocardium proteomic study were observed for proteins regulated by PGC-1α, PPARs and ERRα. These proteins, namely trifunctional enzyme subunit α-complex, short chain acylCoA dehydrogenase, acylCoA thioesterase 2, fatty acid binding protein-3, NADH dehydrogenase (ubiquinone) flavoprotein 2, NADH dehydrogenase (ubiquinone) 1α-subunit 10 and Hspd1 protein, are involved in fatty acid oxidation and mitochondrial respiration. In conclusion, the methyl donor deficiency produces detrimental effects on fatty acid oxidation and energy metabolism of myocardium through imbalanced methylation/acetylation of PGC-1α and decreased expression of PPARα and ERRα. These data are of pathogenetic relevance to perinatal cardiomyopathies.

Availability of pharmacogenetic and pharmacogenomic information in anticancer drug monographs in France: personalized cancer therapy.

AIM: To determine the availability of pharmacogenetic and pharmacogenomic information for healthcare professionals in France during 2009 for anticancer drugs. MATERIALS & METHODS: We searched in the informatic version of the VIDAL dictionary which is currently used by healthcare professionals in France. We then compared this with data available in the PubMed database. RESULTS: Among the 109 anticancer molecules available in France during 2009, 13 have pharmacogenomic or pharmacogenetic information in their monographs. In the scientific literature, we found numerous pharmacogenomic and pharmacogenetic biomarkers concerning 43 of the 109 anticancer agents. Some are pharmacogenomic biomarkers related to drug effectiveness, others are pharmacogenetic biomarkers related to drug toxicity. CONCLUSION: We believe that the lack of pharmacogenomic and pharmacogenetic information in drug monographs reflects the relative newness of the discipline. However, pharmacogenetics and pharmacogenomics can offer valuable information for improving the safety of drugs, reducing toxicity and predicting nonresponders. The drugs might then be incorporated into clinical practice through several strategies, including increased drug labeling and better education of healthcare professionals.