Delta-aminolevulinate Synthase Research Articles

Design of Potential Inhibitors of Pf5-ALAS in Liver Stage Plasmodium falciparum: A Sustainable Chemotherapeutic Approach to Address Antimalarial Resistance

Plasmodium falciparum delta-aminolevulinate synthase (Pf5-ALAS) is the first enzyme in the heme biosynthetic pathway, and it is a liver stage specific enzyme in the developmental stages of Plasmodium falciparum. 8-amino quinoline derivatives have been reported to be active against liver stage parasite and hence was used as a template in the design of 12 derivatives as sustainable chemotherapeutics that were screened in this study designed as potential inhibitors of Pf5-ALAS. The target was modelled due to the unavailability of its experimentally validated 3-dimensional (3D) structure. The binding energy of all 12 designed compounds ranged from -7.9 to -9.1 Kcal/mol which all performed better than primaquine a known inhibitor of liver stage malaria. All twelve designed compounds had comparatively good pharmacokinetic profiles and did not present a toxicity risk, according to in silico ADMET prediction. The position and presence of a functional group that introduces a synergistic impact and subsequently raises the binding energy are highlighted in the qualitative structural assessment of the top three hits. This might pave way to highly economical new antimalarial therapeutic for sustainability health and wellbeing in sub-Saharan Africa and beyond.

Brain tissue haemoglobin expression in saline-perfused vs non-perfused rodents

Haemoglobin beta (Hbb) and delta-aminolevulinate synthase 2 (Alas2) messenger RNA (mRNA) is mainly found in immature red blood cells, reticulocytes, and not in mature erythrocytes. However, these are also expressed in other tissues such as brain cells, mostly neurons. Therefore, exact quantification of neural tissue homogenates may be confounded by remaining blood in the brain vasculature that may give falsely high values of Hbb/Alas2 expression. To investigate and compare the contribution of local Hbb/Alas2 expression, we investigated mRNA expression locally in the hippocampus and prefrontal cortex, in post-sacrifice saline-perfused and non-perfused mice and rats. Although there was a higher level of Hbb/Alas2 transcripts in the non-perfused animals, there was a significant mRNA expression in perfused brains that could at most partially be explained by remaining blood. Finally, we suggest that saline-perfusion should be recommended for quantification of brain Hbb/Alas2 transcripts in homogenates.

Givosiran in acute intermittent porphyria: A personalized medicine approach.

In patients with acute intermittent porphyria (AIP), induction of delta aminolevulinic acid synthase 1 (ALAS1) leads to haem precursor accumulation that may cause recurring acute attacks. In a recent phase III trial, givosiran significantly reduced the attack rate in severe AIP patients. Frequent adverse events were injection-site reaction, fatigue, nausea, chronic kidney disease and increased alanine aminotransferase. To describe the efficacy and safety of givosiran based on a personalized medical approach. We conducted a retrospective patient file study in 25 severe AIP patients treated with givosiran in France. We collected data on clinical and biochemical efficacy along with reports of adverse events. Givosiran drastically reduced the attack rate in our cohort, as 96% were attack-free at the time of the study. The sustained efficacy of givosiran in most patients allowed us to personalize dosing frequency. In 42%, givosiran was only given when haem precursor levels were increasing. Our data suggest that givosiran is most effective when given early in the disease course. We confirmed a high prevalence of adverse events. One patient discontinued treatment due to acute pancreatitis. All patients had hyperhomocysteinemia, and all patients with initial homocysteine levels available showed an increase under treatment. In this context, one patient was diagnosed with pulmonary embolism. The sustained effect of givosiran allowed a decrease in dosing frequency without compromising treatment efficacy. The high prevalence of adverse events emphasizes the importance of restricting the treatment to severe AIP and administering the minimum effective dose for each patient.

Muscle atrophy induced by overexpression of ALAS2 is related to muscle mitochondrial dysfunction

BackgroundALAS2 (delta-aminolevulinate synthase 2) is one of the two isoenzymes catalyzing the synthesis of delta-aminolevulinic acid (ALA), which is the first precursor of heme synthesis. ALAS2-overexpressing transgenic mice (Tg mice) showed syndrome of porphyria, a series of diseases related to the heme anabolism deficiency. Tg mice showed an obvious decrease in muscle size. Muscle atrophy results from a decrease in protein synthesis and an increase in protein degradation, which ultimately leads to a decrease in myofiber size due to loss of contractile proteins, organelles, nuclei, and cytoplasm.MethodsThe forelimb muscle grip strength of age-matched ALAS-2 transgenic mice (Tg mice) and wild-type mice (WT mice) were measured with an automated grip strength meter. The activities of serum LDH and CK-MB were measured by Modular DPP. The histology of skeletal muscle (quadriceps femoris and gastrocnemius) was observed by hematoxylin and eosin (HE) staining, immunohistochemistry, and transmission electron microscope. Real-time PCR was used to detect mtDNA content and UCP3 mRNA expression. Evans blue dye staining was used to detect the membrane damage of the muscle fiber. Single skeletal muscle fiber diameter was measured by single-fiber analyses. Muscle adenosine triphosphate (ATP) levels were detected by a luminometric assay with an ATP assay kit.ResultsCompared with WT mice, the strength of forelimb muscle and mass of gastrocnemius were decreased in Tg mice. The activities of serum CK-MB and LDH, the number of central nuclei fibers, and Evans blue positive fibers were more than those in WT mice, while the diameter of single fibers was smaller, which were associated with suppressed expression levels of MHC, myoD1, dystrophin, atrogin1, and MuRF1. Re-expression of eMyHC was only showed in the quadriceps of Tg mice, but not in WT mice. Muscle mitochondria in Tg mice showed dysfunction with descented ATP production and mtDNA content, downregulated UCP3 mRNA expression, and swelling of mitochondria.ConclusionALAS2 overexpressing-transgenic mice (Tg mice) showed muscle dystrophy, which was associated with decreased atrogin-1 and MuRF-1, and closely related to mitochondrial dysfunction.

Orphan designation: Synthetic double-stranded siRNA oligonucleotide directed against delta-aminolevulinic acid synthase 1 mRNA, covalently linked to a ligand containing three N-acetylgalactosamine residues (givosiran), Treatment of acute hepatic porphyria

Orphan designation: Synthetic double-stranded siRNA oligonucleotide directed against delta-aminolevulinic acid synthase 1 mRNA, covalently linked to a ligand containing three N-acetylgalactosamine residues (givosiran), Treatment of acute hepatic porphyria

Cystathionine \u03b2-synthase (CBS) deficiency suppresses erythropoiesis by disrupting expression of heme biosynthetic enzymes and transporter

The reduced iron usage induced by the suppression of erythropoiesis is a major cause of the systemic iron overload in CBS knockout (CBS−/−) mice. However, the relevant mechanisms are unknown. Here, we examined changes in granulocyte/erythroid cell ratios, iron content, and expression of iron-metabolism proteins, including; two key enzymes involved in the heme biosynthetic pathway, ALAS2 (delta-aminolevulinate synthase 2) and FECH (ferrochelatase), a heme exporter from the cytosol and mitochondria, FLVCR (feline leukemia virus subgroup C cellular receptor) as well as EPO (erythropoietin), EPOR (erythropoietin receptor) and HIF-2α (hypoxia inducible factor-2 subunit α), in the blood, bone marrow or liver of CBS−/− (homozygous), CBS+/− (heterozygous) and CBS+/+ (Wild Type) mice. Our findings demonstrate that CBS deficiency can induce a significant reduction in the expression of ALAS2, FECH, FLVCR, HIF-2α, EPO, and EPOR as well as an increase in interleukin-6 (IL-6), hepcidin and iron content in the blood, bone marrow or liver of mice. We conclude that the suppression of erythropoiesis is mainly due to the CBS deficiency-induced disruption in the expression of heme biosynthetic enzymes and heme-transporter.

A CRISPR/Cas9 Whole-Genome Screen Identifies Genes Required for Aryl Hydrocarbon Receptor-Dependent Induction of Functional CYP1A1.

Environmental pollutants including halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons, including benzo[a]pyrene, exert their deleterious effects through the activation of the aryl hydrocarbon receptor (AHR) and by the resulting transcription of genes not yet fully identified. Ligand-bound AHR translocates from cytoplasm to nucleus, where it dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein. The AHR/ARNT dimer binds to enhancer regions of responsive genes to activate transcription. AHR also mediates carcinogenesis caused by PAHs, likely via CYP1A1, CYP1A2, and CYP1B1, which are massively induced by activated AHR in many tissues and generate carcinogenic electrophilic derivatives of PAHs. In the current study, we have used the mouse GeCKOv2 genome-wide CRISPR/Cas9 library to identify novel genes in the AHR pathway by taking advantage of a B[a]P selection assay that we previously used to identify core AHR pathway genes in Hepa-1c1c7 murine hepatoma cells. Besides Ahr, Arnt, and Cyp1a1, we report the identification of multiple additional putative AHR pathway genes including several that we validated. These include cytochrome P450 reductase (Por), which mediates redox regeneration of cytochromes P450, and 5 genes of the heme biosynthesis pathway: delta-aminolevulinate synthase 1 (Alas1), porphobilinogen deaminase (Hmbs), uroporphyrinogen decarboxylase (Urod), coproporphyrinogen oxidase (Cpox), and ferrochelatase (Fech): heme being an essential prosthetic group of cytochrome P450 proteins. Notably, several of these genes were identified by GeCKO screening, despite not being identifiable by reverse genetics approaches. This indicates the power of high-sensitivity genome-wide genetic screening for identifying genes in the AHR pathway.

Phase 1 Trial of an RNA Interference Therapy for Acute Intermittent Porphyria

BackgroundInduction of delta aminolevulinic acid synthase 1 (ALAS1) gene expression and accumulation of neurotoxic intermediates result in neurovisceral attacks and disease manifestations in patients with acute intermittent porphyria, a rare inherited disease of heme biosynthesis. Givosiran is an investigational RNA interference therapeutic agent that inhibits hepatic ALAS1 synthesis.MethodsWe conducted a phase 1 trial of givosiran in patients with acute intermittent porphyria. In part A of the trial, patients without recent porphyria attacks (i.e., no attacks in the 6 months before baseline) were randomly assigned to receive a single subcutaneous injection of one of five ascending doses of givosiran (0.035, 0.10, 0.35, 1.0, or 2.5 mg per kilogram of body weight) or placebo. In part B, patients without recent attacks were randomly assigned to receive once-monthly injections of one of two doses of givosiran (0.35 or 1.0 mg per kilogram) or placebo (total of two injections 28 days apart). In part C, patients who had recurrent attacks were randomly assigned to receive injections of one of two doses of givosiran (2.5 or 5.0 mg per kilogram) or placebo once monthly (total of four injections) or once quarterly (total of two injections) during a 12-week period, starting on day 0. Safety, pharmacokinetic, pharmacodynamic, and exploratory efficacy outcomes were evaluated.ResultsA total of 23 patients in parts A and B and 17 patients in part C underwent randomization. Common adverse events included nasopharyngitis, abdominal pain, and diarrhea. Serious adverse events occurred in 6 patients who received givosiran in parts A through C combined. In part C, all 6 patients who were assigned to receive once-monthly injections of givosiran had sustained reductions in ALAS1 messenger RNA (mRNA), delta aminolevulinic acid, and porphobilinogen levels to near normal. These reductions were associated with a 79% lower mean annualized attack rate than that observed with placebo (exploratory efficacy end point).ConclusionsOnce-monthly injections of givosiran in patients who had recurrent porphyria attacks resulted in mainly low-grade adverse events, reductions in induced ALAS1 mRNA levels, nearly normalized levels of the neurotoxic intermediates delta aminolevulinic acid and porphobilinogen, and a lower attack rate than that observed with placebo. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov number, NCT02452372.)

Isolation of blue-green eggshell pigmentation-related genes from Putian duck through RNA-seq

BackgroundThe diversity of avian eggshell colour plays important biological roles in ensuring successful reproduction. Eggshell colour is also an important trait in poultry, but the mechanisms underlying it are poorly understood in ducks. This study aimed to provide insights into the mechanism of blue-green eggshell colour generation.ResultsHere, white-shelled ducks (HBR) and blue-green-shelled ducks (HQR) were selected from Putian black ducks, and white-shelled ducks (BBR) were selected from Putian white ducks. Transcriptional changes in the shell gland were analysed using RNA-sequencing on the Illumina HiSeq 2500. Twenty-seven individual cDNA libraries were sequenced and generated an average of 7.35 million reads per library; 70.6% were mapped to the duck reference genome, yielding an average of 13,794 genes detected, which accounted for approximately 86.39% of all 15,967 annotated duck genes. A total of 899 differentially expressed genes (DEGs) were detected between the HQR and BBR groups, and 373 DEGs were detected between the HQR and HBR groups. We analysed the DEGs in the HQR-vs-BBR and HQR-vs-HBR comparisons. None of these DEGs were directly involved in the eggshell pigmentation process in HQR-vs-HBR, while UDP-glucuronosyltransferase 2A2 (UGT2A2) and UDP-glucuronosyltransferase 1–1-like (UGT1–1-like), which participate in biliverdin breakdown, were two of the DEGs in HQR-vs-BBR. In the RT-qPCR results, delta-aminolevulinic acid synthase 1 (ALAS1) and EPRS glutamyl-prolyl-tRNA synthetase were significantly upregulated in the HBR group compared with the HQR and BBR groups (P < 0.05). Haem oxygenase (HMOX1) was significantly downregulated in BBR compared with HQR and HBR (P < 0.05). Biliverdin reductase A (BLVRA), GUSB glucuronidase beta, cytochrome c-type haem lyase, protohaem IX farnesyltransferase and UGT2A2 were significantly upregulated in HBR and BBR compared with HQR (P < 0.05).ConclusionsWe conducted a comparative transcriptome analysis of the shell glands of Putian white ducks and Putian black ducks. None of the differentially regulated pathways were directly involved in the eggshell pigmentation process in the HQR-vs-HBR comparison, while 2 DEGs related to biliverdin breakdown were found in HQR-vs-BBR. Based on the RT-qPCR results, we can speculate that both HQR and HBR can produce biliverdin, but HBR cannot accumulate it. Compared with HQR, BBR produced less biliverdin and did not accumulate it.

Transcriptomic biomarkers of altered erythropoiesis to detect autologous blood transfusion.

Autologous blood transfusion is a powerful means of improving performance and remains one of the most challenging methods to detect. Recent investigations have identified 3 candidate reticulocytes genes whose expression was significantly influenced by blood transfusion. Using quantitative reverse transcription polymerase chain reaction as an alternative quantitative method, the present study supports that delta-aminolevulinate synthase 2 (ALAS2), carbonic anhydrase (CA1), and solute carrier family 4 member 1 (SLC4A1) genes are down-regulated post-transfusion. The expression of these genes exhibited stronger correlation with immature reticulocyte fraction than with reticulocytes percentage. Moreover, the repression of reticulocytes' gene expression was more pronounced than the diminution of immature reticulocyte fraction and reticulocyte percentage following blood transfusion. It suggests that the 3 candidate genes are reliable predictors of bone marrow's response to blood transfusion and that they represent potential biomarkers for the detection of this method prohibited in sports.

Chronic Treatment with Isoniazid Causes Protoporphyrin IX Accumulation in Mouse Liver.

Isoniazid (INH) can cause hepatotoxicity. In addition, INH is contraindicated in patients suffering from porphyrias. Our metabolomic analysis revealed that chronic treatment with INH in mice causes a hepatic accumulation of protoporphyrin IX (PPIX). PPIX is an intermediate in the heme biosynthesis pathway, and it is also known as a hepatotoxin. We further found that INH induces delta-aminolevulinate synthase 1 (ALAS1), the rate-limiting enzyme in heme biosynthesis. We also found that INH downregulates ferrochelatase (FECH), the enzyme that converts PPIX to heme. In summary, this study illustrated that chronic treatment with INH causes PPIX accumulation in mouse liver in part through ALAS1 induction and FECH downregulation. This study also highlights that drugs can disrupt the metabolic pathways of endobiotics and increase the risk of liver damage.

Preclinical Development of a Subcutaneous ALAS1 RNAi Therapeutic for Treatment of Hepatic Porphyrias Using Circulating RNA Quantification.

The acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1) by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs) following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology.

A Novel Hemizygous I418S Mutation in the ALAS2 Gene in a Young Korean Man with X-Linked Sideroblastic Anemia

A Novel Hemizygous I418S Mutation in the ALAS2 Gene in a Young Korean Man with X-Linked Sideroblastic Anemia

A New ALAS2 Mutation Inducing a Male Lethal X-Linked Sideroblastic Anemia

X-linked Sideroblastic Anemia (XLSA, MIM# 300751) is due to mutations in the erythroid-specific form of 5-aminolevulinate synthase (ALAS2) gene. Main features of this condition are microcytic anemia, iron deposits in the mitochondria of erythroid precursors (ring sideroblasts) and an X linked pattern of inheritance. However, up to one third of described cases have been reported in females mainly due to a highly skewed X-chromosome inactivation (Ducamp, Kannengiesser et al. 2011). We report for the first time in a large four generations pedigree a new mutation in ALAS2 gene inducing a Male Lethal X-linked Syndrome ascertained through an adult heterozygous female with a mild form of congenital sideroblastic anemia (CSA).The propositus of this non consanguineous family (Fig 1; individual III;9) was a female from European ancestry. She exhibited a unexplained congenital, non regenerative, macrocytic (MCV 107fL, moderate anemia (Hb 10.4 g/dL), (first assessment at 6 years old). RBC transfusions were required only twice during pregnancy. The diagnosis of CSA was made at 23 years old when the bone marrow aspiration performed, showed 38% of ring sideroblasts. Erythrocyte protoporphyrin concentration was measured in the female proband carrying an ALAS2 mutation. The protoporphyrin concentration was within the normal range of values: 1.6 µmoles/L of red blood cells (less than 1.9 µmoles/L of red blood cells), as previously observed in XLSA cases. The level of serum ferritin was 224ng/ml (N:11-306) and transferrin saturation was 90%. A heterozygote ALAS2 deleterious missense mutation c.622G>T,p.Val208Phe affecting a conserved amino acid was found. A constitutive skewed X-chromosome inactivation was demonstrated as previously reported in affected females with XLSA. However erythroid bone marrow precursor did not exhibited different pattern repartition in term of apoptosis or dyserythropoisesis. Her daughter and her mother exhibited the same mutation but did not have skewed X-chromosome inactivation and were unaffected with a normal blood count. A close inspection of the pedigree confirms a large female predominance (22 females/ 7 males) over four generations (/F/M ratio 3.1). No affected male were identified in the pedigree. Moreover a high level of miscarriage was found only in female carrying the ALAS2 mutation, as shown in the pedigree (Fig. 1). Adding the number of miscarriage (18 over the four generations) to the number of males alive the ratio of M/F over 4 generation is close of 1: 1.04 (24/23). These data highly suggest an X-linked dominant disorder with pre natal male lethality.Our pedigree confirms the non redundant role of the erythroid-specific form of delta-aminolevulinate synthase in foetal hematopoïesis; Moreover our propositus case showed that in case of X-linked Sideroblastic Anemia (XLSA) affected female, a research of excess of miscarriage in the pedigree should be considered and should evocate a male lethal XLSA. This should have an impact in term of genetic counselling. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Octabromodiphenyl ether — porphyrogenicity after repeated administration to rats

Octabromodiphenyl ether (OctaBDE) is a flame retardant which has been withdrawn from common use due to its negative effect on the environment. The literature data regarding its toxicity addresses its effect on liver function, the endocrine and reproductive systems, as well as its developmental toxicology aspects. The aim of this study was to investigate the effect of repeated administration of OctaBDE on heme biosynthesis in rats. The study was performed on female Wistar rats. OctaBDE was administered intragastrically at four different doses (2, 8, 40 or 200 mg/kg/day) for 7, 14, 21 or 28 days. The following measures of heme synthesis disturbance were used: urinary excretion of porphyrins, liver concentration of porphyrins, the activity of delta-aminolevulinate synthase (ALA-S) and delta-aminolevulinate dehydratase (ALA-D) in the liver. After 28 days of exposure, lower ALA-S and ALA-D activity was observed in the liver. Additionally, increased concentrations of high carboxylated porphyrins (octa- and heptacarboxyporphyrins) were found in the liver: from 2- to 10-fold after the 2 mg/kg/day doses and from 4- to 14-fold after the 8-200 mg/kg/day doses. The porphyrogenic effect of OctaBDE was also evidenced by augmented, dose-dependent and exposure time-dependent, concentrations of total porphyrins in urine (2-7.5-fold increase) and their urinary excretion (2-9-fold increase). Tetracarboxyporphyrins predominated in the urine; their concentrations increased 2.5-10 fold. The study revealed that repeated exposure to OctaBDE affects heme biosynthesis and the levels of porphyrins. The lowest effective level which induced changes in porphyrin concentration was 2 mg/kg/day.

Evaluation of mRNA marker specificity for the identification of five human body fluids by capillary electrophoresis

The identification of forensically relevant human body fluids through messenger RNA (mRNA) profiling is of interest to the forensic community. Previous studies have proposed several tissue-specific mRNA markers to achieve this goal. Seven markers for the following genes were selected for evaluation in this study: histatin 3 (HTN3) and statherin (STATH) for saliva, mucin 4 (MUC4) for vaginal secretions, matrix metalloproteinase 7 (MMP7) for menstrual blood, delta-aminolevulinate synthase 2 (ALAS2) for peripheral blood, and protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen. The expression of these markers was examined in each body fluid. All mRNA markers were present in their target body fluids. Peripheral blood and saliva showed little cross-reactivity with the selected markers. However, a high level of cross-reactivity was observed between the vaginal secretion marker MUC4 and saliva stains. Semen showed a high level of cross-reactivity with the selected markers. Co-expression of the predicted body fluid markers was detected in menstrual blood and vaginal secretion stains. The expression pattern of these mRNA markers varied through the menstrual cycle time points tested. Differences in gene expression levels and marker cross-reactivity were observed in the donors tested. Despite the presence of cross-reactivity and co-expression, each of the body fluids examined have distinct gene expression profiles, allowing for body fluid identification based on mRNA profiling.

New Mutation in Erythroid-Specific Delta-Aminolevulinate Synthase as the Cause of X-Linked Sideroblastic Anemia Responsive to Pyridoxine

Background/Aims: Congenital sideroblastic anemias (SA) are characterized by the presence of ringed sideroblasts in the bone marrow. The most common form is X-linked SA, which results from mutations in erythroid-specific δ-aminolevulinate synthase (ALAS2), the first enzyme in heme biosynthesis. In addition, autosomal recessive mutations in the erythroid-specific mitochondrial transporter SLC25A38 and glutaredoxin 5 (GLRX5) have recently been identified in SA patients with isolated erythroid phenotype. Materials and Methods: We studied 5 young males with congenital SA from the Czech Republic. Mutation analysis was performed on the complete coding regions of 3 candidate genes (ALAS2, SLC25A38 and GLRX5), and the enzyme activity of ALAS2 was measured by a continuous spectrophotometric assay. Results: We found the previously published R452H and R452C ALAS2 mutations in 3 patients. A novel K156E substitution in ALAS2 was discovered in 1 pyridoxine-responsive patient. The functional study showed that this substitution severely decreases ALAS2 enzyme activity. In 1 pyridoxine-refractory patient, no mutations were detected in ALAS2, SLC25A38 or GLRX5. Conclusion: Our report extends the list of known ALAS2 mutations, with the addition of a novel K156E substitution that is responsive to pyridoxine treatment and contributes to the general knowledge of congenital SA cases characterized worldwide.

Changes in brain biogenic amines and haem biosynthesis and their response to combined administration of succimers and Centella asiatica in lead poisoned rats

This study was designed to investigate the therapeutic potential of meso 2,3-dimercaptosuccinic acid (DMSA) and one of its monoesters, monoisoamyl DMSA (MiADMSA), individually or when administered in combination with an extract of Centella asiatica against experimental lead intoxication in rats. Biochemical variables indicative of alterations in the central nervous system and haem biosynthesis were investigated to determine the toxicity in male Wistar rats. Thirty five rats were exposed to 0.2% lead acetate for 10 weeks, followed by 10 days of treatment with DMSA and MiADMSA (50 mg kg(-1), i.p., once daily) alone and in combination with C. asiatica (200 mg kg(-1), p.o., once daily). Biochemical variables indicative of oxidative stress and brain biogenic amines, along with lead concentration in blood and brain, were measured. Lead exposure caused a significant depletion of blood and brain delta-aminolevulinic acid dehydratase (ALAD) activity, an important enzyme of the haem biosynthesis pathway, and glutathione (GSH) level. These changes were accompanied by a marked increase in reactive oxygen species (ROS) level, thiobarbituric acid reactive substances (TBARS), delta-aminolevulinic acid synthase (ALAS) and oxidized glutathione (GSSG) activity in blood and brain. Significant depletion of brain noradrenaline (norepinephrine, NE), 5-hydroxytryptamine (5-HT), dopamine (DA) and acetylcholinesterase (AChE) also were observed following lead exposure. Also seen was a significant depletion in brain glutathione peroxidase (GPx), glutathione S-transferase (GST) and monoamine oxidase activity, as well as blood and brain superoxide dismutase (SOD) activity. These biochemical changes were correlated with an increased uptake of lead in blood and brain. Combined administration of MiADMSA and C. asiatica was most effective in reducing these alterations, including biogenic amines, besides reducing body lead burden, compared with individual treatment with MiADMSA. Certain other biochemical variables responded favourably to combination therapy and monotherapy with MiADMSA. Thus, supplementation of C. asiatica during chelation could be recommended for achieving optimum effects of chelation therapy.

Deficiency of MnSOD in Hematopoietic Stem Cells Causes a Sideroblastic Anemia-Like Phenotype.

Abstract 1991Poster Board I-1013 Introduction:Sideroblastic anemia is caused by an inappropriate incorporation of iron into hemoglobin, and as such a phenotype demonstrating ringed sideroblasts, improper iron deposition, and malignancy is observed. The only known hereditary cause of this disease is a mutation in delta-aminolevulinate synthase 2 (ALAS2), which is a mitochondrially located enzyme that catalyzes the first step in heme synthesis, while the causes of the acquired or idiopathic forms remain unknown. With the knowledge that the free radical superoxide (O2•-) is able to displace iron from heme-containing proteins, it is hypothesized that the loss of the mitochondrial anti-oxidant enzyme manganese superoxide dismutase (MnSOD), encoded by the SOD2 gene, may cause disruption of proper heme synthesis and as such may contribute to a sideroblastic anemia-like phenotype. Methods: Due to the early lethality of the constitutive MnSOD knock-out mouse, a floxed (flanked by loxP) MnSOD mouse was created. Homozygous hematopoietic stem cell specific SOD2 knock-outs were generated by breeding the floxed MnSOD mice with mice expressing Cre-recombinase under the control of the hematopoietic stem cell specific promoter Vav. Results: Mice harboring the hematopoietic knock-out of MnSOD are born viable, and are able to live to maturity. Complete blood count (CBC) analysis of knock-out mice demonstrated a decrease in red blood cells (RBC), thymic white blood cells, and splenic white blood cells. The knock-outs additionally showed a decrease in hemoglobin to 5 g/dL compared to 11 g/dL in controls, a reduction in hematocrit to 32% versus 43.5% in controls, and a reticulocyte count of 31.6% compared to only 7.9% in controls. CBC analysis also revealed many RBC morphology flags including hypochromasia, ansiocytosis, and hematocrit variance. These findings were visualized in blood smears (see below). Finally, electron paramagnetic resonance spectrometry of plasma from the knock-out mice showed a ∼2-fold decrease in transferrin levels compared to controls. Conclusions: The loss of MnSOD in the hematopoietic stem cell population of a mouse creates a viable organism, but with an apparent sideroblastic anemia phenotype. Additionally, the white blood cells within these organisms also may be affected due to the decrease in total cellularity, and thus could contribute to immunodeficiency and/or malignancy later in the animal's life. Overall, this phenotype is very comparable to those observed in human patients, and as such MnSOD may play an undiscovered role in acquired or idiopathic sideroblastic anemia. Future work will focus on further characterization of the animals, examination of human blood samples for MnSOD mutations, and the use of SOD mimetics to potentially reverse the observed phenotype. This work was supported by NIH grants CA73612 and CA115438 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

New Mutation in ALAS2 as the Cause of X-Linked Sideroblastic Anemia Responsive to Pyridoxine: Comparison of ALAS2-Defective and DMT1-Defective BFU-E Growth.

Abstract 4051Poster Board III-986Congenital sideroblastic anemias (SA) are hypochromic microcytic anemias with secondary iron overload. Local iron accumulation in mitochondria results in the formation of ringed sideroblasts in the bone marrow. Defects in heme synthesis, Fe-S cluster biogenesis and export have been reported in SA patients. X-linked sideroblastic anemia is a rare gonosomal recessive disorder, resulting from mutations in erythroid-specific delta-aminolevulinate synthase (ALAS2), the first enzyme of heme biosynthesis. It affects mostly males. Few cases of affected females, due to skewed X-inactivation pattern favoring the mutant allele have been reported. Inefficiency of ALAS2 leads to decreased heme production and ineffective erythropoiesis. We investigated a group of 4 young males (average age 28) with congenital SA. Two of the patients had family history of SA and iron overload in males corresponding with the X-linked inheritance. All the patients had hemoglobin level lower than 119 g/L, mean corpuscular volume under 71 fL, increased serum ferritin and numerous sideroblasts in their bone marrow. We performed screening of all exons and promoter region of ALAS2. One patient was negative for ALAS2 mutation, and is a candidate for SLC25A38 mutation screening which only recently has been published as the second most frequent lesion in congenital SA. In two patients we found previously published R452H and R452C mutations; a novel K156E substitution was discovered in one patient. All these three patients responded partially to high dose of pyridoxine. K156 is a conserved amino acid residue and K156E substitution is SIFT predicted as affecting the protein function (the functional study is ongoing). This mutation was not found in 40 healthy controls or 10 patients with myelodysplastic syndrome with SA (RARS, RCMD-RS). Based on our previous study with DMT-1-deficient erythroid progenitors (Priwitzerova et al. Blood 2004;103:3991-2), we also evaluated a possible erythropoietic defect of ALAS2-mutant erythroid progenitors using methylcellulose-based colony assays. We observed that the in vitro growth of all aforementioned ALAS2-mutant patient's erythroid progenitors (BFU-Es) is not affected, which is in contrast to the defective growth of DMT1-mutant erythroid progenitors. These results suggest that impaired heme synthesis is better tolerated by erythroid progenitors than general iron deficiency due to the block in erythroid iron uptake by DMT1 and that the defect in heme synthesis does not fully account for the impaired hemoglobinization and poor growth of DMT1-defective BFU-Es. These data support the role of iron in other processes involved in erythroid-colony development apart from heme synthesis. In conclusion, we present a novel K156E ALAS2 mutation leading to pyridoxine-responsive X-linked sideroblastic anemia. Using BFU-E assays we also submit that lesions in iron-dependent proteins apart from defective heme synthesis contribute to impaired erythroid colony formation in previously described DMT1-mutant erythroid progenitors.Grant support: Ministry of Health Czech Republic grants NS9935-3 and NS10281-3 and Ministry of Education, Youth and Sports program MSM 6198959205. Disclosures:No relevant conflicts of interest to declare.