Elucidating the Role of Human ALAS2 C-terminal Mutations Resulting in Loss of Function and Disease.

The conserved enzyme aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in certain bacteria and eukaryotes by catalyzing the condensation of glycine and succinyl-CoA to yield aminolevulinic acid. In humans, the ALAS isoform responsible for heme production during red blood cell development is the erythroid-specific ALAS2 isoform. Owing to its essential role in erythropoiesis, changes in human ALAS2 (hALAS2) function can lead to two different blood disorders. X-linked sideroblastic anemia results from loss of ALAS2 function, while X-linked protoporphyria results from gain of ALAS2 function. Interestingly, mutations in the ALAS2 C-terminal extension can be implicated in both diseases. Here, we investigate the molecular basis for enzyme dysfunction mediated by two previously reported C-terminal loss-of-function variants, hALAS2 V562A and M567I. We show that the mutations do not result in gross structural perturbations, but the enzyme stability for V562A is decreased. Additionally, we show that enzyme stability moderately increases with the addition of the pyridoxal 5'-phosphate (PLP) cofactor for both variants. The variants display differential binding to PLP and the individual substrates compared to wild-type hALAS2. Although hALAS2 V562A is a more active enzyme in vitro, it is less efficient concerning succinyl-CoA binding. In contrast, the M567I mutation significantly alters the cooperativity of substrate binding. In combination with previously reported cell-based studies, our work reveals the molecular basis by which hALAS2 C-terminal mutations negatively affect ALA production necessary for proper heme biosynthesis.

From chemistry to genomics: A concise history of the porphyrias.

We describe developments in understanding of the porphyrias associated with each step in the haem biosynthesis pathway and the role of individuals whose contributions led to major advances over the past 150 years. The first case of erythropoietic porphyria was reported in 1870, and the first with acute porphyria in 1889. Photosensitisation by porphyrin was confirmed by Meyer-Betz, who self-injected haematoporphyrin. Günther classified porphyrias into haematoporphyria acuta, acuta toxica, congenita and chronica. This was revised by Waldenström into porphyria congenita, acuta and cutanea tarda, with the latter describing those with late-onset skin lesions. Waldenström was the first to recognise porphobilinogen's association with acute porphyria, although its structure was not solved until 1953. Hans Fischer was awarded the Nobel prize in 1930 for solving the structure of porphyrins and the synthesis of haemin. After 1945, research by several groups elucidated the pathway of haem biosynthesis and its negative feedback regulation by haem. By 1961, following the work of Watson, Schmid, Rimington, Goldberg, Dean, Magnus and others, aided by the availability of modern techniques of porphyrin separation, six of the porphyrias were identified and classified as erythropoietic or hepatic. The seventh, 5-aminolaevulinate dehydratase deficiency porphyria, was described by Doss in 1979. The discovery of increased hepatic 5-aminolaevulinate synthase activity in acute porphyria led to development of haematin as a treatment for acute attacks. By 2000, all the haem biosynthesis genes were cloned, sequenced and assigned to chromosomes and disease-specific mutations identified in all inherited porphyrias. These advances have allowed definitive family studies and development of new treatments.

Isoniazid inhibits human erythroid 5-aminolevulinate synthase: Molecular mechanism and tolerance study with four X-linked protoporphyria patients.

Mutations in the C-terminus of human erythroid 5-aminolevulinate synthase (hALAS2), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, are associated with two different blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA-causing mutations yield hALAS2 variants with decreased activity, while XLPP-causing mutations result in a gain-of-function of hALAS2. There are no specific treatments for XLPP. Isonicotinic acid hydrazide (isoniazid, INH), an antituberculosis agent, can cause sideroblastic anemia as a side-effect, by limiting PLP availability to hALAS2, via inhibition of pyridoxal kinase or reaction with pyridoxal to form pyridoxal isonicotinoyl hydrazone. We hypothesized that INH also binds and directly inhibits hALAS2. Using fluorescence-activated cell sorting and confocal fluorescence microscopy, we demonstrate that INH reduces protoporphyrin IX levels in HeLa cells expressing either wild-type hALAS2 or XLPP variants. In addition, PLP and pyridoxamine 5'-phosphate (PMP) reversed the cellular inhibition of hALAS2 activity by INH. Steady-state kinetic analyses with purified hALAS2 indicated that INH directly inhibits the enzyme, noncompetitively or uncompetitively, with an apparent Ki of 1.2µM. Circular dichroism spectroscopy revealed that INH triggered tertiary structural changes in hALAS2 that altered the microenvironment of the PLP cofactor and hampered the association of PLP with apo-hALAS2. Treatment of four XLPP patients with INH (5mg·kg-1·day-1) over a six-month period was well tolerated but without statistically significant modification of PPIX levels. These results, taken together, permit us to further an INH inhibition kinetic mechanism for ALAS, which suggests the possible use of INH-derived drugs in treating patients with XLPP and potentially other protoporphyrin-accumulating porphyrias.

Human Erythroid 5-Aminolevulinate Synthase Mutations Associated with X-Linked Protoporphyria Disrupt the Conformational Equilibrium and Enhance Product Release.

Regulation of 5-aminolevulinate synthase (ALAS) is at the origin of balanced heme production in mammals. Mutations in the C-terminal region of human erythroid-specific ALAS (hALAS2) are associated with X-linked protoporphyria (XLPP), a disease characterized by extreme photosensitivity, with elevated blood concentrations of free protoporphyrin IX and zinc protoporphyrin. To investigate the molecular basis for this disease, recombinant hALAS2 and variants of the enzyme harboring the gain-of-function XLPP mutations were constructed, purified, and analyzed kinetically, spectroscopically, and thermodynamically. Enhanced activities of the XLPP variants resulted from increases in the rate at which the product 5-aminolevulinate (ALA) was released from the enzyme. Circular dichroism spectroscopy revealed that the XLPP mutations altered the microenvironment of the pyridoxal 5'-phosphate cofactor, which underwent further and specific alterations upon succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon binding of ALA to the XLPP variants demonstrated that the protein conformational transition step associated with product release was predominantly affected. Of relevance is the fact that XLPP could also be modeled in cell culture. We propose that (1) the XLPP mutations destabilize the succinyl-CoA-induced hALAS2 closed conformation and thus accelerate ALA release, (2) the extended C-terminus of wild-type mammalian ALAS2 provides a regulatory role that allows for allosteric modulation of activity, thereby controlling the rate of erythroid heme biosynthesis, and (3) this control is disrupted in XLPP, resulting in porphyrin accumulation.

The heme biosynthesis pathway is essential for Plasmodium falciparum development in mosquito stage but not in blood stages.

Heme is an essential cofactor for aerobic organisms. Its redox chemistry is central to a variety of biological functions mediated by hemoproteins. In blood stages, malaria parasites consume most of the hemoglobin inside the infected erythrocytes, forming nontoxic hemozoin crystals from large quantities of heme released during digestion. At the same time, the parasites possess a heme de novo biosynthetic pathway. This pathway in the human malaria parasite Plasmodium falciparum has been considered essential and is proposed as a potential drug target. However, we successfully disrupted the first and last genes of the pathway, individually and in combination. These knock-out parasite lines, lacking 5-aminolevulinic acid synthase and/or ferrochelatase (FC), grew normally in blood-stage culture and exhibited no changes in sensitivity to heme-related antimalarial drugs. We developed a sensitive LC-MS/MS assay to monitor stable isotope incorporation into heme from its precursor 5-[(13)C4]aminolevulinic acid, and this assay confirmed that de novo heme synthesis was ablated in FC knock-out parasites. Disrupting the FC gene also caused no defects in gametocyte generation or maturation but resulted in a greater than 70% reduction in male gamete formation and completely prevented oocyst formation in female Anopheles stephensi mosquitoes. Our data demonstrate that the heme biosynthesis pathway is not essential for asexual blood-stage growth of P. falciparum parasites but is required for mosquito transmission. Drug inhibition of pathway activity is therefore unlikely to provide successful antimalarial therapy. These data also suggest the existence of a parasite mechanism for scavenging host heme to meet metabolic needs.

Molecular and functional analysis of the C-terminal region of human erythroid-specific 5-aminolevulinic synthase associated with X-linked dominant protoporphyria (XLDPP).

Frameshift mutations in the last coding exon of the 5-aminolevulinate synthase (ALAS) 2 gene were described to activate the enzyme causing increased levels of zinc- and metal-free protoporphyrin in patients with X-linked dominant protoporphyria (XLDPP). Only two such so-called gain-of-function mutations have been reported since the description of XLDPP in 2008. In this study of four newly identified XLDPP families, we identified two novel ALAS2 gene mutations, a nonsense p.Q548X and a frameshift c.1651-1677del26bp, along with a known mutation (delAGTG) found in two unrelated families. Of relevance, a de novo somatic and germinal mosaicism was present in a delAGTG family. Such a phenomenon may explain the high proportion of this mutation in XLDPP worldwide. Enhancements of over 3- and 14-fold in the catalytic rate and specificity constant of purified recombinant XLDPP variants in relation to those of wild-type ALAS2 confirmed the gain of function ascribed to these enzymes. The fact that both p.Q548X and c.1651-1677del26bp are located in close proximity and upstream from the two previously described mutations led us to propose the presence of a large gain-of-function domain within the C-terminus of ALAS2. To test this hypothesis, we generated four additional nonsense mutants (p.A539X, p.G544X, p.G576X and p.V583X) surrounding the human XLDPP mutations and defined an ALAS2 gain-of-function domain with a minimal size of 33 amino acids. The identification of this gain-of-function domain provides important information on the enzymatic activity of ALAS2, which was proposed to be constitutively inhibited, either directly or indirectly, through its own C-terminus.

X-linked dominant protoporphyria: a new porphyria.

X-linked dominant protoporphyria (XLDPP) was first reported in the genetics literature in 2008. It has a phenotype very similar to erythropoietic protoporphyria (EPP), but is distinguished from EPP by higher concentrations of erythrocyte protoporphyrin (of which a high proportion is zinc-chelated), its apparently higher incidence of liver disease, and an X-linked dominant pattern of inheritance. Dermatologists should understand how XLDPP differs from EPP, in order to advise newly diagnosed patients correctly about the genetic implications and the long-term management strategy. We present a case series of XLDPP to introduce this condition to the dermatology literature.

Molecular expression and characterization of erythroid-specific 5-aminolevulinate synthase gain-of-function mutations causing X-linked protoporphyria.

X-linked protoporphyria (XLP) (MIM 300752) is a recently recognized erythropoietic porphyria due to gain-of-function mutations in the erythroid-specific aminolevulinate synthase gene (ALAS2). Previously, two exon 11 small deletions, c.1699_1670ΔAT (ΔAT) and c.1706_1709ΔAGTG (ΔAGTG), that prematurely truncated or elongated the ALAS2 polypeptide, were reported to increase enzymatic activity 20- to 40-fold, causing the erythroid accumulation of protoporphyrins, cutaneous photosensitivity and liver disease. The mutant ΔAT and ΔAGTG ALAS2 enzymes, two novel mutations, c.1734ΔG (ΔG) and c.1642C>T (p.Q548X), and an engineered deletion c.1670-1671TC>GA p.F557X were expressed, and their purified enzymes were characterized. Wild-type and ΔAGTG enzymes exhibited similar amounts of 54- and 52-kDa polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas the ΔAT and p.F557X had only 52-kDa polypeptides. Compared to the purified wild-type enzyme, ΔAT, ΔAGTG and Q548X enzymes had increased specific activities that were only 1.8-, 3.1- and 1.6-fold, respectively. Interestingly, binding studies demonstrated that the increased activity Q548X enzyme did not bind to succinyl-CoA synthetase. The elongated ΔG enzyme had wild-type specific activity, kinetics and thermostability; twice the wild-type purification yield (56 versus 25%); and was primarily a 54-kDa form, suggesting greater stability in vivo. On the basis of studies of mutant enzymes, the maximal gain-of function region spanned 57 amino acids between 533 and 580. Thus, these ALAS2 gain-of-function mutations increased the specific activity (ΔAT, ΔAGTG and p.Q548X) or stability (ΔG) of the enzyme, thereby leading to the increased erythroid protoporphyrin accumulation causing XLP.

Clinical and genetic characteristics of congenital sideroblastic anemia: comparison with myelodysplastic syndrome with ring sideroblast (MDS-RS).

Sideroblastic anemia is characterized by anemia with the emergence of ring sideroblasts in the bone marrow. There are two forms of sideroblastic anemia, i.e., congenital sideroblastic anemia (CSA) and acquired sideroblastic anemia. In order to clarify the pathophysiology of sideroblastic anemia, a nationwide survey consisting of clinical and molecular genetic analysis was performed in Japan. As of January 31, 2012, data of 137 cases of sideroblastic anemia, including 72 cases of myelodysplastic syndrome (MDS)-refractory cytopenia with multilineage dysplasia (RCMD), 47 cases of MDS-refractory anemia with ring sideroblasts (RARS), and 18 cases of CSA, have been collected. Hemoglobin and MCV level in CSA are significantly lower than those of MDS, whereas serum iron level in CSA is significantly higher than those of MDS. Of 14 CSA for which DNA was available for genetic analysis, 10 cases were diagnosed as X-linked sideroblastic anemia due to ALAS2 gene mutation. The mutation of SF3B1 gene, which was frequently mutated in MDS-RS, was not detected in CSA patients. Together with the difference of clinical data, it is suggested that genetic background, which is responsible for the development of CSA, is different from that of MDS-RS.

Annular elastolytic giant cell granuloma associated to late-onset X-linked dominant protoporphyria.

X-linked dominant protoporphyria (XLDPP) is a genetic disorder that affects the synthesis of the heme group due to an increase in delta-aminolaevulinate synthase 2 (ALAS2) enzyme activity. Moreover, annular elastolytic giant-cell granuloma (AEGCG) is a rare reactive granulomatous dermatosis, usually associated with actinic damage. An 86-year-old man presented with edematous-erythematous lesions in photoexposed areas of the face and on the dorsum of both hands. Protoporphyrin levels in serum and feces were significantly elevated and a heterozygous frameshift mutation in the exon 11 of the ALAS2 gene: c.1706-1709del (p.Glu569GlyfsX24) was identified. Concomitantly, we observed an annular plaque with raised borders on the back of his right hand, clinically and histologically compatible with a diagnosis of AEGCG. Skin lesions disappeared only upon use of a physical sunscreen. We report two rare photodermatoses in an elderly patient and discuss the significance of dermal elastic fiber damage induced by the XLDPP as a main triggering factor of AEGCG.

Human aminolevulinate synthase structure reveals a eukaryotic-specific autoinhibitory loop regulating substrate binding and product release.

5'-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel mutations of human erythroid-specific isozyme ALAS2, within a C-terminal (Ct) extension of its catalytic core that is only present in higher eukaryotes, lead to gain-of-function X-linked protoporphyria (XLP). Here, we report the human ALAS2 crystal structure, revealing that its Ct-extension folds onto the catalytic core, sits atop the active site, and precludes binding of substrate succinyl-CoA. The Ct-extension is therefore an autoinhibitory element that must re-orient during catalysis, as supported by molecular dynamics simulations. Our data explain how Ct deletions in XLP alleviate autoinhibition and increase enzyme activity. Crystallography-based fragment screening reveals a binding hotspot around the Ct-extension, where fragments interfere with the Ct conformational dynamics and inhibit ALAS2 activity. These fragments represent a starting point to develop ALAS2 inhibitors as substrate reduction therapy for porphyria disorders that accumulate toxic heme intermediates.

Clinical, Biochemical, and Genetic Characterization of North American Patients With Erythropoietic Protoporphyria and X-linked Protoporphyria.

Importance: Autosomal recessive erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are rare photodermatoses presenting with variable degrees of painful phototoxicity that markedly affects quality of life. The clinical variability, determinants of severity, and genotype/phenotype correlations of these diseases are not well characterized. Objective: To describe the baseline clinical characteristics, genotypes, and determinants of disease severity in a large patient cohort with EPP or XLP. Design, Setting, and Participants: A prospective observational study was conducted among patients with confirmed diagnoses of EPP or XLP from November 1, 2010, to December 6, 2015, at 6 academic medical centers of the Porphyrias Consortium of the National Institutes of Health Rare Diseases Clinical Research Network. Detailed medical histories, including history of phototoxicity and treatment, were collected on standardized case report forms. Patients underwent baseline laboratory testing, total erythrocyte protoporphyrin (ePPIX) testing, and molecular genetic testing. Data were entered into a centralized database. Main Outcomes and Measures: Results of biochemical and genetic tests were explored for association with clinical phenotype in patients with EPP or XLP. Results: Of the 226 patients in the study (113 female and 113 male patients; mean [SD] age, 36.7 [17.0] years), 186 (82.3%) had EPP with a FECH (OMIM 612386) mutation and the common low-expression FECH allele IVS3-48T>C, and only 1 patient had 2 FECH mutations. Twenty-two patients had XLP (9.7%; 10 male and 12 female patients), and 9 patients (4.0%) had elevated ePPIX levels and symptoms consistent with protoporphyria but no detectable mutation in the FECH or ALAS2 (OMIM 301300) gene. Samples of DNA could not be obtained from 8 patients. Patients' mean (SD) age at symptom onset was 4.4 (4.4) years. Anemia (107 [47.3%]), history of liver dysfunction (62 [27.4%]), and gallstones (53 [23.5%]) were commonly reported. Higher ePPIX levels were associated with earlier age of symptom onset (median ePPIX levels for those who developed symptoms before vs after 1 year of age, 1744 vs 1567 µg/dL; P = .02), less sun tolerance (median ePPIX levels for those reporting symptoms before vs after 10 minutes of sun exposure, 2233 vs 1524 µg/dL; P ≤ .001), and increased risk of liver dysfunction (median ePPIX levels for those with liver dysfunction vs normal liver function, 2016 vs 1510 µg/dL; P = .003). Patients with EPP and FECH missense mutations had significantly lower ePPIX levels than those with other mutations (1462 vs 1702 µg/dL; P = .01). Male patients with XLP had significantly higher ePPIX levels, on average, than did patients with EPP (3574 vs 1669 µg/dL; P < .001). Marked clinical variability was seen in female patients with XLP owing to random X-chromosomal inactivation. Conclusions and Relevance: These data suggest that higher ePPIX levels are a major determinant of disease severity and risk of liver dysfunction in patients with EPP or XLP. These findings provide a framework for clinical monitoring and management of these disorders.

X-linked dominant protoporphyria: The first reported Japanese case.

A 12-year-old boy with photosensitivity since 3 years of age presented with small concavities on both cheeks, the nasal root and the dorsal surface of both hands. According to the clinical features, erythropoietic protoporphyria (EPP) was suspected. Urine and blood samples were tested for porphyrin derivatives, which revealed a markedly elevated level of erythrocyte protoporphyrin (EP) and a diagnosis of EPP was made. The patient's mother had no photosensitivity, however, lesions appearing slightly as small scars were found on the dorsum of her right hand; his elder sister and father showed no rash. The EP levels were elevated in samples from his mother and mildly elevated in those from his elder sister and father. To obtain a definitive diagnosis, genetic analyses were performed using samples from all family members, which revealed no mutations in the ferrochelatase-encoding gene (FECH), which is responsible for EPP. Instead, a pathological mutation of the 5-aminolevulinic acid synthase-encoding gene (ALAS2) was identified in samples from the patient, his mother and his elder sister, confirming a definitive diagnosis of X-linked dominant protoporphyria (XLDPP). This is the first Japanese family reported to have XLDPP, demonstrating evidence of the condition in Japan. In addition, because XLDPP is very similar to EPP in its clinical aspects and laboratory findings, a genetic analysis is required for the differential diagnosis.

Photosensitivity in the elderly-think of late-onset protoporphyria.

Photosensitivity is the clinical hallmark of both erythropoietic protoporphyria (EPP) and X-linked dominant protoporphyria (XLDPP). Both disorders result from a hereditary dysfunction in heme biosynthesis. Disease onset is usually in early childhood. However, rare patients with late-onset EPP in association with a myeloproliferative disorder or myelodysplastic syndrome have been reported. In this issue, Livideanu et al. describe the first patient with late-onset XLDPP.

Differential gene expression profiling between wild-type and ALAS2-null erythroblasts: identification of novel heme-regulated genes.

To identify erythroid-specific heme-regulated genes, we performed differential expression analysis between wild-type and heme-deficient erythroblasts, which had been prepared from wild-type and erythroid-specific delta-aminolevulinate synthase-null mouse ES cells, respectively. Among 8737 clones on cDNA array, 40 cDNA clones, including 34 unknown ESTs, were first selected by their high expression profiles in wild-type erythroblasts, and evaluated further for their erythroid-lineage specificity, expression in hematopoietic tissues in vivo, and heme-dependent expression, which yielded 11, 4, and 4 genes, respectively. Because of the selection strategy employed, the final 4 were considered as the newly identified erythroid-specific heme-regulated genes. These 4 genes were uncoupling protein 2, nucleolar spindle-associated protein, cellular nucleic acid-binding protein, and a novel acetyltransferase-like protein. These findings thus suggest that heme may regulate a wide variety of hitherto unrecognized genes, and further analysis of these genes may clarify their role in erythroid cell differentiation.

[Acquired, vitamin B6-responsive, primary sideroblastic anemia, an enzyme deficiency in heme synthesis]. / Erworbene, vitamin-B6-responsive, primäre sideroblastische Anämie, ein Enzymdefekt der Hämsythese.

The activity of delta-aminolevulinic acid synthetase (ALAS), the rate-limiting enzyme in heme synthesis, has been found to be markedly reduced (13% of controls) in erythroblasts of a patient with acquired, primary sideroblastic anemia. Administration of vitamin B6 (pyridoxin, 200-600 mg/d) resulted in complete reconstitution of erythroblastic ALAS-activity with concomitant disappearance of all hematologic abnormalities. The findings show that the therapeutic efficacy of pyridoxin in primary sideroblastic anemia is due to its effect on defective ALAS. More generally, the data support the view that almost all features of primary sideroblastic anemia can be ascribed to a disturbance of heme synthesis in erythroblasts.