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The Mendelian inheritance pattern of acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria is autosomal dominant, but the clinical phenotype is heterogeneous. Within the general population, penetrance is low, but among first-degree relatives of a symptomatic proband, penetrance is higher. These observations suggest that genetic factors, in addition to mutation of the specific enzyme of the biosynthetic pathway of heme, contribute to the clinical phenotype. Recent studies by others suggested that the genotype of the transporter protein ABCB6 contribute to the porphyria phenotype. Identifying the molecule(s) that are transported by ABCB6 has been problematic and has led to uncertainty with respect to how or if variants/mutants contribute to phenotypic heterogeneity. Knockout mouse models of Abcb6 have not provided a direction for investigation as homozygous knockout animals do not have a discrete phenotype. To address the proposed link between ABC6 genotype and porphyria phenotype, a large cohort of patients with acute hepatic porphyria and erythropoietic protoporphyria was analyzed. Our studies showed that ABCB6 genotype did not correlate with disease severity. Therefore, genotyping of ABCB6 in patients with acute hepatic porphyria and erythropoietic protoporphyria is not warranted.

As part of the inflammatory response by macrophages, Irg1 is induced, resulting in millimolar quantities of itaconate being produced. This immunometabolite remodels the macrophage metabolome and acts as an antimicrobial agent when excreted. Itaconate is not synthesized within the erythron but instead may be acquired from central macrophages within the erythroid island. Previously, we reported that itaconate inhibits hemoglobinization of developing erythroid cells. Herein we show that this action is accomplished by inhibition of tetrapyrrole synthesis. In differentiating erythroid precursors, cellular heme and protoporphyrin IX synthesis are reduced by itaconate at an early step in the pathway. In addition, itaconate causes global alterations in cellular metabolite pools, resulting in elevated levels of succinate, 2-hydroxyglutarate, pyruvate, glyoxylate, and intermediates of glycolytic shunts. Itaconate taken up by the developing erythron can be converted to itaconyl–coenzyme A (CoA) by the enzyme succinyl-CoA:glutarate-CoA transferase. Propionyl-CoA, propionyl-carnitine, methylmalonic acid, heptadecanoic acid, and nonanoic acid, as well as the aliphatic amino acids threonine, valine, methionine, and isoleucine, are increased, likely due to the impact of endogenous itaconyl-CoA synthesis. We further show that itaconyl-CoA is a competitive inhibitor of the erythroid-specific 5-aminolevulinate synthase (ALAS2), the first and rate-limiting step in heme synthesis. These findings strongly support our hypothesis that the inhibition of heme synthesis observed in chronic inflammation is mediated not only by iron limitation but also by limitation of tetrapyrrole synthesis at the point of ALAS2 catalysis by itaconate. Thus, we propose that macrophage-derived itaconate promotes anemia during an inflammatory response in the erythroid compartment.

During erythroid differentiation, the erythron must remodel its protein constituents so that the mature red cell contains hemoglobin as the chief cytoplasmic protein component. For this, ∼109 molecules of heme must be synthesized, consuming 1010 molecules of succinyl-CoA. It has long been assumed that the source of succinyl-coenzyme A (CoA) for heme synthesis in all cell types is the tricarboxylic acid (TCA) cycle. Based upon the observation that 1 subunit of succinyl-CoA synthetase (SCS) physically interacts with the first enzyme of heme synthesis (5-aminolevulinate synthase 2, ALAS2) in erythroid cells, it has been posited that succinyl-CoA for ALA synthesis is provided by the adenosine triphosphate-dependent reverse SCS reaction. We have now demonstrated that this is not the manner by which developing erythroid cells provide succinyl-CoA for ALA synthesis. Instead, during late stages of erythropoiesis, cellular metabolism is remodeled so that glutamine is the precursor for ALA following deamination to α-ketoglutarate and conversion to succinyl-CoA by α-ketoglutarate dehydrogenase (KDH) without equilibration or passage through the TCA cycle. This may be facilitated by a direct interaction between ALAS2 and KDH. Succinate is not an effective precursor for heme, indicating that the SCS reverse reaction does not play a role in providing succinyl-CoA for heme synthesis. Inhibition of succinate dehydrogenase by itaconate, which has been shown in macrophages to dramatically increase the concentration of intracellular succinate, does not stimulate heme synthesis as might be anticipated, but actually inhibits hemoglobinization during late erythropoiesis.

Differentiating erythroid cells synthesize large quantities of heme for hemoglobinization. While the transcriptional regulation and enzymatic mechanisms of the heme synthetic enzymes are well characterized, we lack mechanistic understanding of how their protein stability, cofactor incorporation and functional interactions with mitochondrial housekeeping proteins are regulated. These mechanisms can rapidly alter the rate of heme synthesis in response to external stimuli and metabolic requirements, and are critical for heme regulation within a tissue-specific and developmental context. CLPX, a mitochondrial protein unfoldase best understood for its function in a proteasome-like enzyme complex with the peptidase CLPP (the CLPXP ATP-dependent protease) plays a central role in regulation of mitochondrial protein turnover, is one such heme regulatory protein. CLPX activates yeast ALAS, which catalyzes the committed step of the heme synthesis pathway, by facilitating the incorporation of its cofactor, PLP, and is required for erythroid heme synthesis in zebrafish (Kardon et al. Cell 2015). Paradoxically, it regulates the turnover of ALAS1 and ALAS2 protein in vertebrate cell lines and appears to regulate the heme synthesis downstream of ALAS (Kubota et al. JBC 2016, Yien et al. PNAS 2017). However, it is not known if vertebrate ALAS was activated by ALAS, or if the requirement for CLPX in vertebrate heme synthesis was caused its regulation of ALAS activity (Figure A). To dissect the roles of CLPX in erythroid heme synthesis, we knocked out Clpxand Clpp in murine erythroleukemia (MEL) cells and assayed the activity, stability, and steady state levels of the heme synthesis enzymes, ALAS2 and FECH, which colocalize with ALAS in the mitochondrial matrix. Consistent with previous observations, Clpx -/- MEL cells had a heme defect, while Clpp -/-cells did not (Figure B). However, in contrast to previous observations in the yeast model, CLPX is not required for ALAS activation in erythroid cells, but plays a key role in regulating ALAS2 turnover in concert with the CLPP peptidase (Figure C). During erythroid differentiation, CLPP protein levels are decreased, stabilizing ALAS2 protein (Figure D). Although differentiating Clpx -/-and Clpp -/- MEL cells did not demonstrate any changes in ALAS2 turnover, likely because steady-state levels of CLPP protein were already decreased (Figure E), we observed an increase in steady-state ALAS2 protein levels and a dramatic increase in ALAS2 enzyme activity. In vitro mitochondrial iron transport/heme synthesis assays revealed a heme defect in Clpx -/-MEL cells, suggesting that CLPX plays a role in mitochondrial iron metabolism. Collectively, these data suggest a complex, differentiation-stage specific regulation of heme synthesis by the CLPXP proteolytic complex (Figure F). As Clpx -/- mouse embryos die by about E9.5 (mousephenotype.org), we dissected the in vivo role of Clpxin erythropoiesis by analyzing the phenotypes of clpxa and clpxb mutant zebrafish obtained from ZIRC. To accomplish this, we crossed clpxa and clpxb mutant zebrafish into Tg(lcr:GFP) zebrafish line in which erythroid cells are fluorescently labeled with GFP (Ganis et al Dev Biol 2012). We observed that clpxa mutant zebrafish had an early erythropoietic defect at 24 hpf that resolved at 48hpf; this developmental defect was not observed in clpxbmutant zebrafish. Benzidine staining of heme in mutant zebrafish revealed that while clpxa was dispensable for erythroid heme synthesis, clpxb was required for erythroid hemoglobinization (Figure G). Lastly, clpxbzebrafish mutants continued to be developmentally delayed and did not survive past 5 dpf. Collectively, our observations in cell lines and in the zebrafish model demonstrate that Clpx is essential for the maintenance of differentiated erythroid cells, as well as for the differentiation of the erythroid lineage. The control of heme synthesis and erythroid development by CLPX reveals how mitochondrial physiology and heme synthesis are interdependent. Our results reveal an important regulatory node where the mitochondrial protein quality control machinery intersects with key steps in heme synthesis. Further, our studies provide important genetic tools for dissecting these regulatory components in isolation as well as within the in vivocontext of erythropoiesis. Figure Disclosures No relevant conflicts of interest to declare.

Congenital erythropoietic porphyria (CEP) is an autosomal recessive disorder of heme synthesis characterized by reduced activity of uroporphyrinogen III synthase and the accumulation of nonphysiologic isomer I porphyrin metabolites, resulting in ineffective erythropoiesis and devastating skin photosensitivity. Management of the disease primarily consists of supportive measures. Increased activity of 5-aminolevulinate synthase 2 (ALAS2) has been shown to adversely modify the disease phenotype. Herein, we present a patient with CEP who demonstrated a remarkable improvement in disease manifestations in the setting of iron deficiency. Hypothesizing that iron restriction improved her symptoms by decreasing ALAS2 activity and subsequent porphyrin production, we treated the patient with off-label use of deferasirox to maintain iron deficiency, with successful results. We confirmed the physiology of her response with marrow culture studies.

Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well understood. To better understand these regulatory mechanisms, we profiled gene expression in EPO-treated fetal liver cells to identify novel iron regulatory genes (Figure A). We determined that FAM210B, a mitochondrial inner membrane protein, was essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation (Figure B). Fam210b deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur cluster formation (Figure C). These defects were corrected with a lipid-soluble small molecule iron transporter in Fam210b-deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that FAM210B is not a mitochondrial iron transporter, but is required for optimal mitochondrial iron import during erythroid differentiation (Figure D). FAM210B is also required for optimal FECH activity in differentiating erythroid cells. As FAM210B interacts with the terminal enzymes of the heme synthesis pathway, we propose that FAM210B functions as an adaptor protein to facilitate the formation of an oligomeric mitochondrial iron transport complex, which is required for the increase in iron acquisition for heme synthesis during terminal erythropoiesis (Figure E). Collectively, our data reveal a novel mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism. Figure. Figure. Disclosures Palis: Rubies Therapeutics: Consultancy.

Hepatic siderosis is common in patients with porphyria cutanea tarda (PCT). Mutations in the hereditary hemochromatosis (hh) gene (HFE) explain the siderosis in approximately 20% patients, suggesting that the remaining occurrences result from additional genetic and environmental factors. Two genes known to modify iron loading in hh are hepcidin (HAMP) and hemojuvelin (HJV). To determine if mutations in or expression of these genes influenced iron overload in PCT, we compared sequences of HAMP and HJV in 96 patients with PCT and 88 HFE C282Y homozygotes with marked hepatic iron overload. We also compared hepatic expression of these and other iron-related genes in a group of patients with PCT and hh. Two intronic polymorphisms in HJV were associated with elevated serum ferritin in HFE C282Y homozygotes. No exonic polymorphisms were identified. Sequencing of HAMP revealed exonic polymorphisms in 2 patients with PCT: heterozygosity for a G→A transition (G71D substitution) in one and heterozygosity for an A→G transition (K83R substitution) in the other. Hepatic HAMP expression in patients with PCT was significantly reduced, regardless of HFE genotype, when compared with patients with hh but without PCT with comparable iron overload. These data indicate that the hepatic siderosis associated with PCT likely results from dysregulated HAMP.

GNPAT (chromosome 1q42.2) encodes the peroxisomal enzyme glyceronephosphate O-acyltransferase. In a previous study, DNA of men with hemochromatosis and HFE p.C282Y homozygosity and either markedly increased iron stores or normal or mildly increased iron stores were evaluated with exome sequencing. Positivity for the GNPAT polymorphism p.D519G (rs11558492) was significantly greater in men with markedly increased iron stores (McLaren CE et al., Hepatology 2015;62:429-39). This result suggests that the p.D519G is a candidate modifier of iron phenotypes in p.C282Y homozygotes. To learn more, we examined associations of p.D519G, age, iron-related variables, and daily alcohol consumption with iron stores in p.C282Y homozygotes classified by extremes of iron overload phenotypes. We defined markedly increased iron stores as serum ferritin >1000 µg/L and either hepatic iron >236 µmol/g dry weight or mobilizable iron >10 g by induction phlebotomy (men and women). Normal or mildly elevated iron stores were defined as serum ferritin There were 56 participants (53 men, 3 women), of whom 41 (38 men, 3 women) had markedly increased iron stores and 15 others had normal or mildly increased iron stores (all men). The mean age of the 56 participants was 55 ± 10 (SD) y. Prevalences of swollen/tender 2nd/3rd metacarpophalangeal joints and elevated serum levels of aspartate or alanine aminotransferase were significantly greater in participants with markedly increased iron stores. Only participants with markedly increased iron stores had cirrhosis proven by biopsy. Odds ratios of having markedly increased iron stores for each of the six variables, as determined by univariate logistic regression, are displayed in Table 1. In the multivariable analysis, p.D519G positivity was the only exposure variable significantly associated with markedly increased iron stores (odds ratio 9.9, 95% CI [1.6, 60.3], p = 0.0126). Area under the curve for the multivariable logistic regression analysis was 0.82. We conclude that GNPAT p.D519G is strongly associated with markedly increased iron stores in p.C282Y homozygotes after correction for age, iron-related variables, and daily alcohol consumption. It remains unknown whether p.D519G directly enhances iron transport into the blood by absorptive enterocytes, indirectly augments iron absorption by suppressing hepcidin, or is linked to a putative iron absorption promoter on chromosome 1q. Disclosures No relevant conflicts of interest to declare.

Acute hepatic porphyria (AHP) is a family of rare metabolic disorders including acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), and variegate porphyria (VP), caused by a deficiency in one of the eight enzymes required for heme biosynthesis in the liver. When ALA synthetase (ALAS1), the first and rate limiting step in the pathway, is induced by triggers such as exposure to certain drugs or fasting, the neurotoxic heme intermediates aminolevulinic acid (ALA) and porphobilinogen (PBG) can accumulate upstream of the deficient enzyme leading to acute and potentially life threatening neurovisceral attacks in AHP patients. RNA interference is a naturally occurring cellular mechanism mediated by small interfering RNA (siRNA) that allows for the inhibition of protein synthesis through the cleavage and degradation of a specific mRNA. ALN-AS1 is an investigational RNAi therapeutic that targets ALAS1 in order to decrease ALA and PBG levels and subsequent porphyria attacks. We are currently conducting a phase 1, multinational, randomized, placebo-controlled, study in 3 parts; Part A single ascending dose (SAD), Part B multiple ascending dose (MAD) and Part C multiple dose (MD) study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics (i.e. changes in ALA, PBG and circulating ALAS1 mRNA levels) of subcutaneously administered ALN-AS1 in AIP patients. Part C also has exploratory analyses of clinical activity, including the impact of ALN-AS1 on porphyria attacks, acute healthcare visits, and heme treatment (ClinicalTrials.gov Identifier: NCT02452372). We previously presented interim SAD data demonstrating that ALN-AS1 was generally well tolerated with no serious adverse events (SAEs) or clinically significant laboratory abnormalities related to study drug, and no discontinuations due to AEs. Circulating ALAS1 mRNA levels had a mean (SEM) maximal reduction of 44% ± 8% relative to baseline (p ≤ 0.01 compared to placebo), with concomitant mean (SEM) maximal reductions in ALA and PBG of 77% ± 7% and 73% ± 6%, respectively (p= 0.03 and 0.06 compared to placebo, respectively) at the 0.35 mg/kg dose. In addition, changes in circulating ALAS1 mRNA were highly correlated with changes in urinary ALA and PBG (R2=0.82, p Disclosures Sardh: Alnylam Pharmaceuticals: Consultancy. Harper:Alnylam Pharmaceuticals: Consultancy. Balwani:ICGG Gaucher Registry (Sanofi Genzyme sponsored): Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Honoraria, Speakers Bureau; Alnylam Pharmaceuticals: Consultancy. Anderson:Alnylam Pharmaceuticals: Consultancy. Bloomer:Alnylam Pharamceuticals: Consultancy. Bissel:Alnylam Pharmaceuticals: Consultancy. Desnick:Alnylam Pharmaceuticals: Consultancy. Bonkovsky:Alnylam Pharamceuticals: Consultancy. Penz:Alnylam Pharmaceuticals: Employment, Equity Ownership. Chan:Alnylam Pharmaceuticals: Employment, Equity Ownership. Soh:Alnylam Pharmaceuticals: Employment, Equity Ownership. Querbes:Alnylam Pharmaceuticals: Employment, Equity Ownership. Simon:Alnylam Pharmaceuticals: Employment, Equity Ownership. Rees:Alnylam Pharmaceuticals: Consultancy.

Inherited and acquired factors have been implicated in the pathogenesis of porphyria cutanea tarda (PCT), a disorder characterized by a photosensitive dermatosis and hepatic siderosis. This study, comprising 108 patients with PCT, was intended to define the role of hemochromatosis gene (HFE) mutations in the expression of PCT and to determine the contribution of acquired factors including alcohol, hepatitis C virus (HCV), and estrogen. The 2 known HFE mutations, cysteine 282 tyrosine (Cys282Tyr) and histidine 63 asparagine (His63Asp), were detected by polymerase chain reaction, and anti-HCV immunoglobulin G was detected serologically. Liver biopsies were graded for iron content, inflammation, and fibrosis. Estimates of alcohol and estrogen use were based on a questionnaire. Of the PCT patients tested, 19% were homozygous for the Cys282Tyr mutation; controls were equal to 0.5%. The compound heterozygous genotype was detected in 7% of the PCT patients; controls were less than 1%. The transferrin saturation, serum ferritin, and liver iron burden of all PCT patients were higher than those of nonporphyric controls. The highest values were found in PCT patients homozygous for the Cys282Tyr mutation. Of the patients studied, 59% were HCV positive (compared with 1.8% of the population), and 46% consumed more than 70 g of alcohol daily. Of the female patients, 63% were ingesting estrogens. Hepatic damage was most marked in patients with the Cys282Tyr/Cys282Tyr genotype who had HCV and drank heavily. Homozygosity for the Cys282Tyr mutation and HCV are the greatest risk factors for expression of PCT, and in most patients, more than 1 risk factor was identified. It was common for patients with HCV to consume alcohol. Patients with PCT should be screened for HFE mutations and for HCV. (Blood. 2000;95:1565-1571)

Iron-regulatory proteins (IRP1 and IRP2) are RNA-binding proteins that bind to stem-loop structures known as iron-responsive elements (IREs). IREs are located in the 5′- or 3′-untranslated regions (UTRs) of specific mRNAs that encode proteins involved in iron homeostasis. The binding of IRPs to 5′ IREs represses translation of the mRNA, whereas the binding of IRPs to 3′ IREs stabilizes the mRNA. IRP1 and IRP2 binding activities are regulated by intracellular iron levels. In addition, nitric oxide (NO.) increases the affinity of IRP1 for IREs. The role of NO. in the regulation of IRP1 and IRP2 in rat hepatoma cells was investigated by using the NO.-generating compound S-nitroso-N- acetylpenicillamine (SNAP), or by stimulating cells with multiple cytokines and lipopolysaccharide (LPS) to induce NO. production. Mitochondrial and IRP1 aconitase activities were decreased in cells producing NO(.). NO. increased IRE binding activity of IRP1, but had no effect on IRE binding activity of IRP2. The increase in IRE binding activity of IRP1 was coincident with the translational repression of ferritin synthesis. Transferrin receptor (TfR) mRNA levels were increased in cells treated with NO.-generating compounds, but not in cytokine- and LPS-treated cells. Our data indicate that IRP1 and IRP2 are differentially regulated by NO. in rat hepatoma cells, suggesting a role for IRP1 in the regulation of iron homeostasis in vivo during hepatic inflammation.

Symptoms of porphyria cutanea tarda (PCT) resolve when iron stores are depleted by phlebotomy, and a sequence variant of HFE (C282Y) that increases iron absorption by reducing hepcidin expression is a risk factor for PCT. These observations suggest that PCT is an iron dependent disease and that factors that affect iron homeostasis influence the risk of developing the PCT. Recently, a polymorphic variant (D519G) of GNPAT was shown to be enriched in male patients with type I hereditary hemochromatosis (HFE C282Y homozygotes) who presented with a high iron phenotype [McLaren CE, et al. Hepatology Aug;62(2):429-39 2015]. Available evidence suggests that like HFE C282Y, GNPAT D519G increases iron absorption by reducing expression of hepcidin. Therefore, we investigated the prevalence GNPAT D519G in patients with PCT. The study population consisted of 247 patients with PCT. High-resolution DNA melting analysis and Taqman SNP assays were used to identify HFE (C282Y) and GNPAT (D519G) allelic variants, respectively and mutations in uroporphyrinogen decarboxylase (UROD) were identified by nucleotide sequencing. Patients with mutant UROD were categorized as familial PCT (F-PCT) (n=87, 36.0%) and those with wild-type UROD were categorized as sporadic PCT (S-PCT) (n=155, 64.0%). GNPAT D519G was significantly enriched in the patient population (prevalence 22.1%, p Disclosures No relevant conflicts of interest to declare.

Red cells synthesize large quantities of heme during terminal differentiation. Central to erythropoiesis is the transport and trafficking of iron within the cell. Despite the importance of iron transport during erythroid heme synthesis, the molecules involved in intracellular trafficking of iron are largely unknown. In a screen for genes that are up-regulated during erythroid terminal differentiation, we identified FAM210B, a predicted multi-pass transmembrane mitochondrial protein as an essential component of mitochondrial iron transport during erythroid differentiation. In zebrafish and mice, Fam210b mRNA is enriched in differentiating erythroid cells and liver (fetal and adult), which are tissues that require large amounts of iron for heme synthesis. Here, we report that FAM210B facilitates mitochondrial iron import during erythroid differentiation and is essential for hemoglobin synthesis. Zebrafish are anemic when fam210b is silenced using anti-sense morpholinos (Fig. A). CRISPR knockout of Fam210b caused a heme synthesis defect in differentiating Friend murine erythroleukemia (MEL) cells. PPIX levels in Fam210b deficient cells are normal, demonstrating that Fam210b does not participate in synthesis of the heme tetrapyrrole ring. Consistent with this result, supplementation of Fam210b deficient MEL cells with either aminolevulinic acid, the first committed substrate of the heme synthesis pathway or a chemical analog of protoporphyrin IX failed to chemically complement the heme synthesis defect. While Fam210b was not required for basal housekeeping heme synthesis, Fam210b deficientcells showed defective total cellular and mitochondrial iron uptake during erythroid differentiation (Fig. B). As a result, Fam210b deficient cells had defective hemoglobinization. Supplementation of Fam210b-/- MEL cells with non-transferrin iron chelates restored erythroid differentiation and hemoglobin synthesis; whereas, similar chemical complementation could not be achieved in the Tmem14c-/- cells, which have a primary defect in tetrapyrrole transport. (Fig. C). Our findings reveal that FAM210B is required for optimal mitochondrial iron import during erythroid differentiation for hemoglobin synthesis. It may therefore function as a genetic modifier for mitochondriopathies, anemias or porphyrias. Figure 1. Figure 1. Disclosures Bauer: Biogen: Research Funding; Editas Medicine: Consultancy. Orkin:Editas Inc.: Consultancy.

Congenital methemoglobinemia due to cytochrome b5 reductase (CYB5R3) deficiency are sporadic worldwide, although clusters of this disorder have been identified in certain ethnic groups. Here we describe the molecular basis of type I methemoglobinemia due to CYB5R3 deficiency in four patients representing three distinct ethnic backgrounds. Three subjects were of Indian ethnicity one being phenotypically normal and his two cousins diagnosed with type I methemoglobinemia. Causative mutations in the globin genes were excluded after sequencing. A fourth subject, a 12 year old Mexican child with Turner’s syndrome and apparent congenital cyanosis was referred to us for evaluation. Her methemoglobin level was elevated and erythrocyte cytochrome b5 reductase was reduced (4% of the normal range). The fifth individual, a 36 year old woman of Greek ancestry, had a past history of childhood cyanosis and low O2 saturation (82%) post-surgery in 2007. In early 2008 she was diagnosed with methemoglobinemia due to dyspnea, disproportionate fatigue, and significant increase in methemoglobin levels (10%). Genomic DNA from all 5 subjects was isolated, and all nine exons of the CYB5R3 gene were PCR amplified and sequenced. A non-synonymous heterozygous mutation (G>A transversion) in exon 6 resulting in the change of amino acid 178 from alanine to threonine (A178T) was identified in both affected Indian siblings and not in their unaffected cousin. This mutation which is located in the NADH binding domain and in close proximity to the adenosine moiety, and may influence binding of the NADH coenzyme had been previously reported (Dekker et al., 2001). A novel homozygous mutation in an evolutionarily conserved position located in exon 3 (C>G transition) resulting in substitution of serine 53 for arginine (S53R) was found in the child of Mexican descent. This mutation, located within the FAD binding domain, is hypothesized to cause disruption of hydrogen bonding and steric hindrance due to the bulkier arginine side chain, consequently leading to protein instability. Five heterozygous mutations were detected in the subject with Greek ancestry. A synonymous G>A substitution (Pro44Pro) in exon 2, a previously reported type II mutation G>A (Val253Met) and two 3′-UTR single nucleotide polymorphisms (A>G at 29690 nt, and C>A at 29914 nt), all in exon 9, were found in a cis configuration. A novel heterozygous compound mutation in exon 6, a T>G transversion, resulting in substitution of phenylalanine 157 for cysteine (F>C) was identified in the same subject with Greek ancestry (Figure 1). PCR amplified CYB5R3 transcripts harboring either wild-type, S53R, or F157C sequences were cloned, in frame with the 10X histidine tag, into the pET16b expression vector. Sequence fidelity and in frame fusion of all constructs was verified by sequencing from both strands. Recombinant protein was purified using 1 mL niquel-affinity columns. Kinetic studies using recombinant CyB5R3 enzyme will follow. Figure 1. Location of mutation found in 4 cases of congenital type I methemoglobinemia. Figure 1. Location of mutation found in 4 cases of congenital type I methemoglobinemia.

Porphyria cutanea tarda (PCT) is caused by a uroporphomethene (UPN) inhibitor of uroporphyrinogen decarboxylase (Phillips, JD et al, Proc Natl Acad Sci U S A. 2007 104(12):5079–84). In the absence of the inhibitor very little uroporphyrin (URO) is secreted in the urine and the excreted URO is predominantly the isomer-III (URO-III). In contrast, in patients with PCT and mice with experimental PCT the predominant porphyrin excreted in urine is uroporphyrin-I (URO-I). Direct measurements of uroporphyrinogen III synthase activity (UIIIS) in mice with PCT have shown no reduction in activity indicating that an alternate mechanism is responsible. We hypothesized that oxidation of a single bridge carbon of HMB, the product of the porphobilinogen deaminase reaction, is the initial event leading to the porphomethene. Our structural data indicate that oxidized HMB cannot serve as a substrate for UIIIS (Schubert, HL et al, Biochem J. 2008 47(33):8648–55) but can non-enzymatically cyclize to form UPN I. UPN I would then serve as the inhibitor of uroporphyrinogen decarboxylase but and then be fully oxidized to yield URO-I. To test this hypothesis we generated authentic UPN’s I and III. URO’s I and III were reduced with palladium on carbon to yield uroporphyrinogens (Uro’gen) I and III. UPN’s I and III were then generated by controlled photo-oxidation of the corresponding Uro’gens. We then utilized LC-MS to separate and characterize the synthesized UPN’s. Retention times of the two compounds differed by approximately 1 min (UPN III elutes prior to UPN I) but the profile of the daughter ions derived from the UPN’s (molecular mass 835 Da) was identical. The same approach was applied to clarified liver cytosol from mice with experimental PCT. The retention time of the 835 Da UPN identified in mouse liver cytosol corresponded to that of the synthesized UPN I and yielded identical daughter ions. These data support the hypothesis that oxidation of HMB leads to formation of UPN I in vivo. This is concordant with our view that UPN I serves both as the inhibitor of URO-D and as the source of the excess URO-I found in PCT.

A higher than age-expected DNA methylation index is predictive for early disease progression in patients with CLL (Yu et al. Leukemia Research 2006). We hypothesized that miRNA expression can also be silenced by promoter hypermethylation in CLL. Thus, methyl pool or DNA methyltransferase inhibitors can upregulate microRNAs with tumor suppressor characteristics by restoring the “normal” pattern of methylation. Results of patients treated with cladribine, a methyl substrate inhibitor, or 5-azacitidine, a DNA methyltransferase inhibitor were compared in 2 separate clinical protocols. The global DNA methylation decreased after treatment with cladribine or 5-azacitidine in 60% of the patients. At the 2006 ASH meeting, we reported on the consistent upregulation of miR-17-3p, miR-21, miR-29a, miR-29b, miR-29c, miR-30e, miR-104, miR-126, miR-128a, miR-130a, miR-141, miR-142-3p, miR-148a, miR-151, miR-199a, miR-199a*, and miR-301 by real-time PCR using the Early Access Human Panel from Applied Biosystems. Data from two patients on the cladribine protocol and 10 patients on the 5-azacitidine protocol were used for statistical analyses. Non-parametric Wilcoxon tests as well as t-tests were performed. Comparisons were made of the responders versus non-responders, and of cladribine versus 5-azacytidine. Since many of the miRNAs showed differences in the cladribine versus 5-azacytidine comparison, a second responder vs non-responder analysis was performed in which the two cladribine subjects were removed. MiR-195 was statistically more upregulated in the cladribine treated responder whereas miR-29c was statistically most upregulated in the 5-azacitidine treated patients (p=0.02). In the patients with global demethylation after treatment, upregulation of microRNA-195 was observed and directly correlated with regional demethylation of the CpG island, confirmed by bisulfite sequencing. Some of the predicted targets of miR-195 include bcl-2, CNOT6L, USP15, PADAH1B1, and ESRRG. MiR-195 may also have tumor suppressor characteristics as it also targets basic fibroblast growth factor (FGF-2), a gene important in CLL angiogenesis. There has been some evidence suggesting FGF-2 is an oncogene. For example, overexpression of FGF-2 isoforms facilitates growth of NIH 3T3 cells in low serum media and also mediates radioresistance of HeLa cells. FGF-2 is also protective against irradiation activation of p53 in the leukemia cells derived from patients with CLL. Although cladribine has been reported to downregulate FGF-2 by inhibiting adenosine deaminase, downregulation of FGF-2 at the transcript and protein levels was also observed in before and after treatment samples from patients treated with 5-azacytidine. We propose an alternative mechanism by which the FGF-2 transcript is degraded after binding to excess miR-195. In patients responsive to treatment with DNA methylation inhibitors, a regional decrease in the methylation status of the CpG island 5′ to miR-195 may lead to increased expression.

SOCS-1, suppressor of cytokine signaling, is a negative regulator of the JAK2/STAT signal transduction pathway. This pathway, often deregulated in myeloproliferative disorders, has not yet been implicated in chronic lymphocytic leukemia (CLL). We demonstrated SOCS-1 upregulation and miR-214 downregulation with clinical response in a patient treated with cladribine. Similar changes were not seen in a treated patient with progressive disease. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate expression of other genes by complementary pairing at the seed sequence in the 3′-UTR of the target mRNA. Some patients with CLL reportedly regulate bcl-2 expression by downregulating miR-15a and miR-16-1. Although no experimentally verified targets have been demonstrated for miR-214, complete inhibition of miR-214 by anti-sense inhibitors prevents apoptosis in Hela cells. Of the 44 predicted human miRNAs that target SOCS-1 (http://microrna.sanger.ac.uk), we determined the expression of 24 of these by real-time PCR using the Early Access Human Panel from Applied Biosystems. MiR-214 was the only downregulated miRNA after treatment. The other 23 miRNAs were either upregulated or unchanged. We demonstrated SOCS-1 protein upregulation by two fold after cladribine in the “responsive” patient without a concomitant increase in the transcript level as measured by real time PCR. SOCS-1 expression is often silenced by DNA promoter methylation in hematologic and solid tumor malignancies. We report another mechanism of regulation through miRNA translational repression. This suggests involvement of the JAK2/STAT pathway in B-cell survival in CLL.

Chronic lymphocytic leukemia is a disease with a variable outcome. Since early treatment does not result in a survival benefit, clinicians often wait for evidence of progressive disease before offering systemic chemotherapy. We recently reported on the contribution of DNA methylation to disease progression. CLL patients with higher than age-expected global DNA methylation demonstrated a requirement for chemotherapy within 12 months as compared to patients with lower than age expected levels. We hypothesized that treatment of patients with early Rai Stage CLL can delay progression of disease by re-expression of tumor suppressor genes. We used two different DNA methylation inhibitors: a DNA methyltransferase enzyme inhibitor, 5-azacytidine, and a methyl substrate inhibitor, 2-chloro-2-deoxyadenosine. We demonstrate increased microRNA expression in six patients treated with 5-azacytidine and the one patient treated with 2-chloro-2-deoxyadenosine. In 6 out of 7 patients, DNA methylation globally decreased by 8% after treatment. However, consistent microRNA upregulation was seen in mir-17-3p, mir-21, mir-29a, mir-29b, mir-29c, mir-30e, mir-104, mir-126, mir-128a, mir-130a, mir-141, mir-142-3p, mir-148a, mir-151, mir-199a, mir-199a*, and mir-301 by real-time PCR using the Early Access Human Panel from Applied Biosystems. Using the University of California Santa Cruz genome database, mir-17, mir-126, mir-128a, mir-148a, mir-151, mir-199a, and mir-301 are sequence conserved in at least 5 mammalian species and are associated with a CpG island upstream from the predicted transcriptional start site. All but mir-148a are embedded within a known gene. No changes were seen in bcl-2, p53, mir-15, or mir-16. Overexpression of mir-199-a and mir-199a* has been shown to induce cell cycle arrest. Mir-17-3p and mir-21 have anti-apoptotic properties. MiRNAs in patients with CLL are likely regulated by DNA promoter methylation.

We have developed a method to clonally mark hematopoietic stem and lymphoid progenitor cell populations using a novel sequence tag approach. A library containing an 11-base random sequence tag is cloned into a lentivirus vector, packaged using the VSV-G glycoprotein and HIV-1 capsid, and transduced into freshly isolated mouse hematopoietic stem cell (Thy-1.1lowc-kithigh) or progenitor cell (Thy-1.1negc-kithigh) populations. To minimize artifacts introduced by prolonged culture, we have utilized a 3-hour spinoculation protocol performed in the absence of cytokines. Transduction efficiency was evaluated in vitro by methylcellulose colony assay and liquid cultures, and in vivo by transplanting the transduced cells into lethally irradiated mice. A bicistronic lentivirus vector with a CMV promoter driving expression of a transcript encoding Thy-1.2-IRES-GFP was used to optimize the transduction protocol. Liquid culture assays demonstrated 57% transduction efficiency after 5 days of growth, based on expression of the Thy-1.2 and GFP reporter proteins. Mice transplanted with transduced Thy-1.1negc-kithigh progenitor cells were sacrificed after 16 days, a time at which we have previously observed robust progenitor cell engraftment in the thymus while progeny of Thy-1.1lowc-kithigh HSC have not yet appeared. In 4 of 4 transplanted mice, we observed donor-derived cells in the bone marrow, lymph nodes and thymus. The percentage of total cells expressing the lentivirus-derived transgene ranged from 1.6% of bone marrow cells to 20% of thymocytes. Peripheral blood from mice transplanted with transduced HSC were analyzed and monitored every 4 weeks for transgene expression. We observed that although the Thy-1.2 marker was expressed and maintained up to 14 weeks after HSC transplant, GFP transgene expression was minimal. Based on these preliminary results, we have engineered a new lentivirus vector containing random sequence tags and the Thy-1.2 marker. This strategy provides a simple and efficient way of tracking the progeny of individual cells within a transplanted population, using PCR amplification of the random tags found within mature cell populations derived from the transduced cells. Sequence analysis of individual clones derived from different lineages of cells will enable us to better define the lineage potentials of specific progenitor cell subpopulations.

Congenital erythropoietic porphyria (CEP), a rare photomutilating disorder, is due to homozygosity or compound heterozygosity for mutant alleles encoding uroporphyrinogen III synthase (U3S), a cytosolic enzyme that converts hydroxymethyl bilane to uroporphyrinogen III. Deficiency of U3S results in an excess of biologically useless uroporphyrin I (URO I), which causes the photomutilation. Clinical expression of CEP is usually associated with co-existence of congenital dyserythropoietic anemia type I, also an autosomal recessive trait. We evaluated a 3-year-old boy of English-French extraction with a photosensitive bullous dermatosis; CEP was suspected. The boy had a hypochromic, microcytic anemia and thrombocytopenia from birth with hemoglobin values averaging 7.0 g/dL, MCV’s averaging 67 fL and platelet counts averaging 70,000/μL. A physical exam revealed scars on the face, hands and forearms, generalized hirsutism and splenomegaly. The diaper fluoresced under a Wood’s lamp. Fifty mL of urine contained 2,003 μg of URO (normal=trace), 92% of which was URO I. Erythrocyte U3S activity was 21% of controls, confirming the diagnosis of CEP. Red cell morphology was compatible with thalassemia and fluorescent red cells were apparent. Hb electrophoresis revealed 36.3% Hb A, 2.4% Hb A2, 59.5% Hb F and 1.8% of a fast moving peak, likely Hb Bart’s. Both parents were hematologically normal, had no evidence of porphyria, and had normal Hb electrophoreses. Genomic U3S was characterized (including the promoter), no mutations or deletions were found in the child or the parents. Characterization of the α and β-globin loci also revealed no mutations or deletions. Lack of a molecular explanation for either the low U3S activity or the β-thalassemia/thrombocytopenia phenotype led us to sequence GATA1, an X-linked transcription factor common to globin genes and the first 4 steps of the heme biosynthetic pathway in erythrocytes. A mutation was found at codon 216 in the child and on one allele of his mother, changing arginine to tryptophan (R216W). A more conservative arginine to glutamine mutation (R216Q) previously described by Yu et al. (Blood2002; 100:2040–5) is the only known human mutation of the DNA-binding domain of the N-finger of GATA1, and did not result in porphyria. Here, in contrast to the reduced activity of U3S, erythrocyte activities of the proximal heme biosynthetic enzymes were normal. To explore the mechanism responsible for the extremely high Hb F levels we sequenced the proximal promoter regions of the γ globin loci. The Xmn I polymorphism (−158 C to T), associated with increased production of G-γ chains, was identified in the promoter region of one G-γ allele in the child and in one allele of his father. The G-γ to A-γ ratio in the child’s Hb F was 2:1, a moderate increase in the expected ratio. Xmn I, when present with the Hb S mutation, results in variable Hb F elevations but almost always

Global DNA hypomethylation is observed in chronic lymphocytic leukemia, but methylation has not been correlated with clinical outcome. Although patient survival correlates with factors such as the mutational status of the immunoglobulin variable genes, karyotype abnormalities, Zap-70, and CD38 expression, none of these predictors have altered the way clinicians practice. We report interim results in the assessment of global DNA methylation as a predictor of aggressive disease in patients with chronic lymphocytic leukemia. Fourteen patients with chronic lymphocytic leukemia donated blood samples for DNA studies at the same time as blooddraws for their physician visits. All the treatments occurred within one year and the follow-ups were at least within 12 months except for one patient. We thus classified patients into two groups: those who required treatment within one year and those who did not. The cutoff in methylation level providing the smallest observed prediction error was 4.125%; it correctly predicted 5/6 patients not needing treatment within a year and 5/6 patients needing treatment. These observed classification rates were adjusted for the bias resulting from the optimal selection of the cutoff using bootstrap. The adjusted sensitivity and specificity were 74% and 80%, respectively. Asymptomatic patients with chronic lymphocytic leukemia tended to have lower levels of global DNA methylation (median 3.5%) compared to symptomatic patients (median 4.5%). In other words, high levels of global DNA methylation were associated with higher disease burden, corresponding with higher lymphocyte and white blood cell numbers. Five patients without immediate need for cytoreductive therapy were enrolled on a pilot treatment trial with low-dose cladribine, by subcutaneous injection. Three out of the five patients have had a clinical response, a secondary endpoint. Two of the patients have achieved a partial response, as defined by the NCI sponsored working group, with at least a three month follow-up after discontinuation of the drug. Of the two patients with stable or progressive disease on cladribine, their global DNA methylation levels were higher, correlating with more chemotherapy resistant disease. | Age | Sex | %5-MedC | Zap-70 | CD-38 | Rai Stage | FISH | Req Treatment (mos) | |:---------------------------------------------------------------------------- | --- | ------- | ------------ | ------------ | --------- | ------------------------------------------------ | ------------------- | | FISH= fluorescence in situ hybridization; Zap-70 assessed by immunochemistry | | 51 | M | 5.045 | positive | positive | 4 | Del13q14 | 0.75 | | 73 | F | 4.865 | positive | not assessed | 4 | not assessed | 12+ | | 52 | F | 4.665 | positive | negative | 2 | Del13q14 | 2 | | 57 | M | 4.62 | negative | negative | 4 | Del13q14 | 2 | | 66 | M | 4.59 | positive | not assessed | 4 | Del13q14 (5/05) and Del 17p and Del 13q14 (7/05) | 0.25 | | 67 | M | 4.14 | positive | positive | 4 | Trisomy 12 | 10 | | 47 | M | 4.055 | negative | negative | | not assessed | 12+ | | 59 | M | 3.9 | negative | negative | 2 | 46XY | | | 72 | M | 3.54 | negative | not assessed | | not assessed | 8+ | | 64 | M | 3.55 | positive | not assessed | 1 | Del13q14 | 12+ | | 70 | F | 3.47 | not assessed | negative | 4 | Trisomy 12 | 12+ | | 68 | F | 3.47 | positive | negative | 2 | not assessed | 12+ | | 77 | M | 3.2 | not assessed | not assessed | | not assessed | 12+ | DNA methylation Levels in Patients with Chronic Lymphocytic Leukemia ![Figure][1] DNA Methylation Levels in patients with Chronic Lymphocytic Leukemia [1]: pending:yes

CPO, an essential enzyme in the heme biosynthetic pathway, catalyzes the oxidative decarboxylation of two propionate side chains of coproporphyrinogen III to form vinyl groups in the product protoporphyrinogen IX. CPO mutations are responsible for the disease hereditary coproporphyria. All eukaryotes express a highly conserved, oxygen-dependant form of CPO. Yeast and human CPO have a sequence identity of 52% at the amino acid level. To probe the biochemical basis of catalytic activity and to determine the deleterious effects of clinically identified mutations, we determined the crystal structure of CPO from S. cerevisiae. This is the first reported structure of a eukaryotic CPO. A cDNA encoding CPO (Hem13p) was expressed in E. coli as a histidine tagged protein. Hem13p was purified and concentrated to 25 mg/ml and crystals were grown in the presence of 18% PEG 8000, 0.1 M HEPES, pH 7.5, 2% isopropanol, 0.2 M Na-acetate. The crystal structure was determined by optimized sulfur anomalous scattering and refined to a resolution of 2.0 Å. The protein folds into a novel structure featuring a central flat seven-stranded antiparallel beta-sheet flanked on each side with helices. The homodimeric structure of CPO is formed by a short isolated strand that forms a beta-ladder between the two monomers. Each monomer contains an active site formed between the flat beta-sheet and adjacent helices near the dimer interface. Many of the conserved residues are located at this interface. The deep active site cleft is lined by conserved residues and has been captured in an open and a closed conformation in two different crystal forms. The substrate cavity is completely buried in the closed conformation by an approximate 8 Å movement of a helix that forms a lid over the active site. The volume of the enclosed cavity precisely accommodates a modeled molecule of coproporphyrinogen III. The model indicates binding of two propionate side chains of coproporphyrinogen with two invariant arginines. The pyrrole nitrogens of the substrate are positioned to be coordinated by an invariant asparagine in a fashion similar to that described for uroporphyrinogen decarboxylase (EMBO J, 2003, 22: 6225–33). Nineteen point mutations have been described in association with hereditary coproporphyria and we have mapped these to the crystal structure. Only two mapped to the active site. Fourteen mutations were predicted to destabilize the protein and 4 of these occur at the dimer interface. Three mutations mapped to the solvent exposed surface of CPO. The crystal structure supports a model in which the enzymatic reaction occurs in an isolated cavity formed by a conformational change induced by substrate binding. The conformational change could create a binding site for molecular oxygen, the cofactor, and provide a mechanism to protect coproporphyrinogen III from inappropriate oxidation and the subsequent peroxidation of protoporphyrinogen IX.