Advances in understanding the pathogenesis of congenital erythropoietic porphyria

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1 review Advances in understanding the pathogenesis of congenital erythropoietic porphyria Elena Di Pierro, Valentina Brancaleoni and Francesca Granata U.O. di Medicina Interna, Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico, Milano, Italy Summary Congenital erythropoietic porphyria (CEP) is a rare genetic disease resulting from the remarkable deficient activity of uroporphyrinogen III synthase, the fourth enzyme of the haem biosynthetic pathway. This enzyme defect results in overproduction of the non-physiological and pathogenic porphyrin isomers, uroporphyrin I and coproporphyrin I. The predominant clinical characteristics of CEP include bullous cutaneous photosensitivity to visible light from early infancy, progressive photomutilation and chronic haemolytic anaemia. The severity of clinical manifestations is markedly heterogeneous among patients; and interdependence between disease severity and porphyrin amount in the tissues has been pointed out. A more pronounced endogenous production of porphyrins concomitant to activation of ALAS2, the first and rate-limiting of the haem synthesis enzymes in erythroid cells, has also been reported. CEP is inherited as autosomal recessive or X-linked trait due to mutations in UROS or GATA1 genes; however an involvement of other causative or modifier genes cannot be ruled out. Keywords: congenital erythropoietic porphyria, uroporphyrinogen III synthase, haemolytic anaemia, UROS, GATA1 and ALAS2 genes. Congenital erythropoietic porphyria [CEP; Online Inheritance in Man (OMIM) ] is one of the least common porphyrias a group of disorders resulting from deficient activity of a specific enzyme in the haem biosynthetic pathway (Fig 1). Congenital erythropoietic porphyria is also known as G unther s disease, deriving its name from the physician who first recognized it as an inborn error of metabolism and proposed the first significant classification of porphyrias by dividing them in the acute and chronic forms (Madan et al, 2007). Congenital erythropoietic porphyria has been included in the chronic porphyria group and is an Correspondence: Elena Di Pierro, PhD, Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico, U.O. di Medicina Interna, via F. Sforza 35, Milano 20122, Italy. s: elena.dipierro@policlinico.mi.it; elena.dipierro@unimi.it extremely rare disease; its prevalence has been estimated at 1 or less in In the literature, there are approximately 350 case reports, but many of them are duplicated, limiting the actual number of CEP patients to approximately 280. Up to 1997, 130 cases were registered (Fritsch et al, 1997) and 123 more new cases have been reported between 1997 and 2015 (Table SI), excluding the 29 European cases reported in 2012 (Katugampola et al, 2012). Until now no clear racial or sexual predominance has been noted, even if a higher number of affected cases belong to certain restricted areas and consanguineous marriage. Congenital erythropoietic porphyria also occurs in animals such as fox squirrel (Levin & Flyger, 1971), hedgehog (Wolff et al, 2005), rat (Rivera & Leung, 2008), cat (Clavero et al, 2010) and cattle (McAloon et al, 2015). Congenital erythropoietic porphyria was the first human porphyria to be described and related to disturbance of the porphyrin metabolism (Moore, 2009). The first description of CEP was published in 1874 by Schultz as a special form of leprosy, pemphigus leprosus. Subsequently, in , Gunther described the disease in detail and defined it as haematoporphyria congenital (Madan et al, 2007). Although the chemical nature of porphyrin accumulation was extensively studied between 1916 and 1930, it was not until 1969 that the specific enzymatic defect was identified (Romeo & Levin, 1969). In the mid 1950s, Schmid and co-workers underlined the erythroid nature of this disease suggesting porphyria erythropoietica as a more appropriate name (Schmid et al, 1954, 1955). Later, this form began to be called congenital erythropoietic porphyria to reflect an early onset of this clinical status compared with the other erythropoietic forms. Biochemical background Congenital erythropoietic porphyria is an inherited defect in haem biosynthesis caused by a deficient, though not completely absent activity (<1 to ~10% of normal) of the cytosolic enzyme, uroporphyrinogen III synthase (UROS; EC 42175). All efforts to generate a knockout mouse model of CEP were unsuccessful demonstrating that a residual UROsynthase activity is indispensable for embryonic viability (Bensidhoum et al, 1998). Haem biosynthesis is carried out ª 2016 John Wiley & Sons Ltd First published online 11 March 2016 doi: /bjh.13978

2 Fig 1. Human porphyrias and enzymatic defects. Each form of porphyria originates from a specific enzymatic defect. The porphyrias highlighted in green are characterized by prevalent neurological acute symptomatology. The porphyrias highlighted in red are characterized by chronic cutaneous photosensitivity. The variegate, hereditary coproporphyria and harderocoproporphyria present both neurological and cutaneous symptomatology. In hepatocytes, the ALAS1 gene is down regulated by haem. In erythropoietic cells, ALAS2 gene regulation is closely related to both iron and haem homeostasis. ALAS, d-aminolevulinate synthase; ALAD, aminolevulinate dehydratase; PBGD, porphobilinogen deaminase; UROS, uroporphyrinogen III synthase; UROD, uroporphyrinogen decarboxylase; CPOX, coproporphyrinogen oxidase; PPOX, protoporphyrinogen oxidase; FECH, ferrochelatase. by a series of sequential reactions catalysed by eight enzymes. d-aminolevulinate synthase (ALAS), the first enzyme in the pathway, is the rate-limiting step in haem synthesis. In hepatocytes, ALAS1 isoenzyme undergoes feedback inhibition by haem. In erythropoietic cells, ALAS2 regulation is closely related to both iron and haem homeostasis (Ponka, 1997) (Fig 1). UROS is the fourth enzyme in the haem biosynthetic pathway that rearranges and cyclises the linear hydroxymethylbilane (HMB), to form the III isomeric form of uroporphyrinogen (uroporphyrinogen III). A fraction of hydroxymethylbilane escapes from the action of UROS resulting in a non-enzymatic conversion to uroporphyrinogen I isomer (Dailey, 1997). Although uroporphyrinogen I can undergo decarboxylation by uroporphyrinogen decarboxylase (UROD) to form hepta-, hexa- and pentacarboxyl porphyrinogen I and, finally, coproporphyrinogen I, further metabolism cannot proceed because the next enzyme in the pathway, coproporphyrinogen oxidase (CPOX), is stereospecific for the III isomer (Romeo & Levin, 1969), (Fig 2). Under normal physiological conditions, little uroporphyrinogen I isomer is formed because UROS catalytic activity is significantly higher than the other preceding enzymes in the biosynthetic pathway (ALAS, aminolevulinate dehydratase [ALAD] and porphobilinogen deaminase [PBGD, also termed hydroxymethylbilane synthase, HMBS]), thus favouring a predominant synthesis of uroporphyrinogen III over the isomer I (Thunell, 2000). The structure of UROS UROS has been purified from several sources: Euglena gracilis, rat liver, and Escherichia coli (Desnick & Astrin, 2002). Human erythrocyte and recombinant URO-synthase have also been purified and characterized. The cytosolic enzyme has been shown to function as a monomer with a molecular weight of 29 kda (Tsai et al, 1987; Omata et al, 2004). A common characteristic of these enzymes is their instability against heat treatment. The crystal structure of the recombinant human UROS has been determined and the protein has been shown to fold into a two-domain structure, with each domain consisting of a parallel b-sheet surrounded by a-helices and connected with each other by a two-strand anti-parallel b-ladder. Evidence for interdomain flexibility has been reported and the inherent flexibility has been proposed to be important for the substrate binding and product release in the catalytic cycle (Mathews et al, 2001). On the basis of nuclear magnetic resonance (NMR) chemical shift perturbations of the specific residues, the active site has been mapped in the cleft region between structural domains 1 and 2, where most of the 366 ª 2016 John Wiley & Sons Ltd

3 Fig 2. The pathway of haem biosynthesis Starting from hydroxymethylbilane, the green arrows indicate the physiological enzymes and intermediates of the pathway. The blue arrows indicate the non-enzymatic conversion to uroporphyrinogen I. Uroporphyrinogen decarboxylase catalyses both uroporphyrinogen isoforms to subsequent coproporphyrinogen 7-, 6- and 5-COOH representing the hepta-, hexa- and pentacarboxylporphyrin intermediates, respectively. As the next enzyme, coproporphyrinogen oxidase, is only specific to isomer III, coproporphyrinogen I is not catalysed further. conserved residues cluster. However, no evidence for a complex formation has been found (Cunha et al, 2008). The active conformation of UROS is thermodynamically unstable but it remains folded long enough to exert its function. A strong interdependency has been observed between the volume of the side chain of the specific residues and the folded protein (ben Bdira et al, 2014). Experiments in both human cell lines and a murine model have shown that the protein is rapidly eliminated, via the proteosomal instead of the lysosomal pathway (Fortian et al, 2011; Blouin et al, 2013). However additional degradation pathways acting on the eukaryotic intracellular homeostasis of this enzyme have also been suggested (ben Bdira et al, 2014). The activity of UROS The specific action of uroporphyrinogen III synthase during the enzymatic reaction is the inversion of one of the four heterocyclic rings present in the substrate. The ring closure with a concurrent flipping of the D ring produces a molecule whose acetate and propionate side chains are asymmetric with respect to the centre of the molecule (Fig 3). The mechanism of this inversion was investigated extensively by Battersby and co-workers using chemically synthesized analogues of the substrate and intermediate. They demonstrated formation of a spirocyclic pyrrolenine intermediate in the course of the D ring flip and proposed the reaction scheme (Battersby et al, 1976, 1978; Leeper, 1994). This mechanism has been supported by high-level quantum mechanical calculations on model systems of this enzyme (Silva & Ramos, 2008). They suggested that the D-ring of the hydroxymethylbilane substrate binds to the enzyme in a conformation that shields its terminal portion from reacting with the ring A and prevents the formation of biologically non-functional uroporphyrinogen I. As the asymmetry is not restored in the subsequent biosynthetic pathway, all the natural and physiological porphyrins and their derivatives bear asymmetric side chains. The metabolic defect A deficit in UROS activity leads to accumulation of its substrate, hydroxymethylbilane (HMB), most of which is nonenzymatically converted to uroporphyrinogen I, which, in turn, is partially metabolized to coproporphyrinogen I. Given that they do not undergo further reactions leading to haem, both accumulate in large amounts leading to the pathogenic condition by undergoing auto-oxidation to the corresponding porphyrins, uroporphyrin I (UROI) and coproporphyrin I (COPROI), respectively. The accumulation of porphyrins occurs primarily in bone marrow erythroid precursor, where an uninterrupted production of haem is required to produce haemoglobin, and consequently in circulating red blood cells. The accumulated porphyrins are then released from the erythroid cells into plasma by haemolysis or diffusion, deposited in tissues mainly in skin and bones and excreted in large amount in urine (both uro- and coproporphyrins) and via the hepatobiliary route in faeces (chiefly coproporphyrin). Porphyrin overload is also found in spleen, consequent to the high rate of red blood cells destruction. To a lesser extent, porphyrins also accumulate in liver where the haem synthetic machinery is regulated in response to the metabolic requirements (Schmid et al, 1954, 1955). In addition, incidences of porphyrin ª 2016 John Wiley & Sons Ltd 367

4 Fig 3. Enzymatic reaction. Porphobilinogen deaminase [PBGD, Enzyme commission (EC) number 25161], the third enzyme of the pathway, condenses four molecules of porphobilinogen (PBG) successively in a head-to-tail fashion forming a linear tetrapyrrole, hydroxymethylbilane (HMB). Next, uroporphyrinogen III synthase (UROS) converts the HMB into a circular uroporphyrinogen III (URO III). During the enzymatic reaction, D ring of the linear HMB experiences an inversion of configuration. The non-enzymatic ring closure forming uroporphyrinogen I (URO I) preserves the symmetry of side chains. Ac, acetate group; Pr, propionate group. deposition in the meninges, brain, heart, lungs, kidney and intestine have also been reported (Bhutani et al, 1974). The porphyrins are photoreactive intermediates, can absorb long ultraviolet and visible light energy and pass to a higher excited-state. They show a major absorption peak especially between 400 and 410 nm, along with four additional absorption bands of low intensity between 500 and 700 nm (Dawe et al, 2002). The membrane and other cellular components are oxidatively damaged by excited electron transfer to oxygen and free-radical formation or by direct electron transfers between excited porphyrins and the targets. Photo-oxidation leads to loss of membrane integrity and function, disruption of cellular organelles and cell death (Poh-Fitzpatrick, 1998). Clinical symptoms The age at onset and clinical severity are highly variable in CEP patients. Extremely severe forms, starting during pregnancy, are dominated by severe haemolytic anaemia responsible for foetal hydrops and death in utero (Pannier et al, 2003). In contrast, adult late-onset forms exhibit a mild phenotype often restricted to a mild cutaneous photosensitivity (Berry et al, 2005), and associated with myelodysplasia (Kontos et al, 2003) or thrombocytopenia. In the majority of cases, the onset is between the neonatal period and 10 years, and the phenotypic expression of the disease is dominated by skin photosensitivity, starting early in childhood in addition to the haematological complications including neonatal jaundice, transfusion-dependent haemolytic anaemia and/or thrombocytopenia and splenomegaly. However, clinical heterogeneity is a common finding in CEP and phenotypic variability has also been reported among patients in the same family (Ged et al, 2004). A close relationship between the metabolic disturbance reflected by porphyrin excess and the severity of disease expression has been established (Freesemann et al, 1997). Increased ALAS2 activity has been shown to adversely modify the disease phenotype through the increase of porphyrin production (To-Figueras et al, 2011). Dermatological involvement A massive porphyrin accumulation in the skin induces a phototoxic oxygen-dependent damage characterized by subepidermal blistering with little or no dermal inflammatory infiltrate (Poh-Fitzpatrick, 1998). The more or less rapid development of cutaneous reaction depends on the intracellular distribution of the accumulated porphyrins, which in turn is a function of their physicochemical properties. The hydrophilic porphyrins, predominantly accumulated in CEP, are more effective in attacking cytosolic targets instead of mitochondria and other lipid-rich cellular structures, causing a delayed phototoxic reaction. Because of the latency and the insidious development of the reaction, a CEP patient often 368 ª 2016 John Wiley & Sons Ltd

5 fails to realise a connection between the irradiation event and the skin reaction (Thunell, 2000). Histopathological analysis in CEP showed hyperorthokeratosis, flattening of the dermoepidermal junction, diffuse sclerosis in the dermis, and almost total disappearance of sweat and sebaceous glands. Certain features, such as thickened vascular walls, hyalin deposits and elastic fibre fragmentation, were visible in few areas (Arunachalam et al, 2013). The severity of the skin manifestations varies considerably among CEP patients and is dependent on porphyrin amount in the tissue and the degree of light exposure (Pandey et al, 2013). In most cases of CEP, the lifelong cutaneous photosensitivity, induced especially by light of the wavelengths around 405 nm, usually starts in early infancy and is manifested by increased skin fragility and rapid development of vesicles and bullae on hands, face and other sun-exposed areas. Blistering over the entire exposed body surface is reported in severely affected infants as a result of radiation during phototherapy used to treat the neonatal jaundice that mostly occurs in such cases (Harada et al, 2001; Baran et al, 2013). A case of blister formation at pulse oximeter site was also reported, suggesting that a constant exposure at red ( nm) and infrared ( nm) light is enough to induce skin damage (Hogeling et al, 2011). The skin may be thickened, with areas of hypo- and hyperpigmentation. Hypertrichosis of the face and extremities is often prominent. The formed vesicles are prone to be ruptured and might get infected, leading to extensive ulcerations, erosions and scarring. Scarring alopecia of the scalp and eyebrows can occur. Repetitive skin damage, secondary infection and bone resorption produce epidermal atrophy, sclerodermoid changes and significant mutilations as well as shortening and contractures of the digits and limb and loss of facial features (Poh-Fitzpatrick, 1986). Facial disfigurement resulting from destruction of auricular and nasal cartilages, cheeks, lips and forehead can be severe. Fortunately, most cases exhibit marked, but less dramatic lesions (Figs 4 6). Adult-onset patients have milder clinical symptoms and often exhibit only the skin manifestations of the disease (Kontos et al, 2003; Guo et al, 2014). An incidence of malignancy in CEP is rare, which can be attributed to the fact that porphyrins may sensitize the malignant skin cells and subsequent sunlight exposure may result in the elimination of these cells (Pandhi et al, 2003). Haematological manifestations The haematological symptoms range from mild asymptomatic microcytic anaemia to severe transfusion-dependent haemolytic anaemia or pancytopenia. A reduced red cell survival time and an ineffective erythropoiesis as the pathogenic factors of the haemolytic anaemia are found in most patients with CEP. The excessive porphyrin concentrations in late erythroid precursors, reticulocytes and erythrocytes were described to induce fragility with osmotic haemolysis (Fritsch et al, 1997). The presence of the needle-like cytoplasmatic inclusions in the reticulocytes and nucleated red cells indicated formation of the crystallized porphyrins (Merino et al, 2006). Marrow mononuclear cells from CEP patients, cultured under conditions optimizing erythroid differentiation, were reported to reveal fewer percentages of fully matured cells and higher absolute numbers of apoptotic cells than normal cells (Egan et al, 2015). Peripheral blood smears of CEP patients showed presence of morphological abnormalities of erythrocytes (anisocytosis and poikilocytosis) and an increased circulating concentration of the nucleated red blood cells, both reticulocytes and normoblasts (polychromasia). Bone marrow examination demonstrated a condition of dyserythropoiesis combining erythroid hyperplasia and ineffective erythropoiesis. Abnormal normoblasts showed nuclear inclusion bodies containing haemoglobin (Katugampola et al, 2012). The occurrence of haemolysis is supported by elevations in serum unconjugated bilirubin, potassium, lactate dehydrogenase (LDH) and faecal urobilinogen, and undetectable or low levels of serum haptoglobin and haemopexin (Fritsch et al, 1997). Although haemolysis is usually present, it is not always accompanied by anaemia, as the hypertrophied and hyperactive marrow is sometimes able to compensate for the increased rate of erythrocyte destruction. However, the degree of compensation varies over time, leading to exacerbations and remissions of haemolysis and anaemia. On the other hand, cases of anaemia without haemolysis (De et al, 2013; Pandey et al, 2013) or cases restricted to skin photosensitivity have also been reported (Bari, 2007; To-Figueras et al, 2007; Darwich et al, 2011). Secondary splenomegaly develops in response to the increased uptake of abnormal or damaged erythrocytes from the circulation and liver enlargement and porphyrin-rich gallstones can be found in some patients. The longstanding anaemia can result in pancytopenia accompanied by purpura and epistaxis (Fritsch et al, 1997). Non-transfusion dependent iron overload accompanying anaemia can develop in the patients (Lange et al, 1995; Fritsch et al, 1997). In a few particular cases accompanied by thrombocytopenia, increased levels of fetal haemoglobin are displayed in Hb electrophoresis test (Phillips et al, 2007; Di Pierro et al, 2015). Ten patients with a disease onset after the age of 50 years and having an associated myelodysplastic syndrome have been described. In these cases porphyrin concentrations were lower than those in the childhood onset; and the normal erythroid UROS activity was consistent with the fact that the porphyric defect can be restricted to only a minority of hematopoietic cells. The cause that leads to this acquired form of CEP remains to be elucidated (Kontos et al, 2003; Sarkany et al, 2011) and the possibility of considering this latter condition as an erythropoietic porphyria secondary to myeloid malignancy rather than a late-onset of congenital CEP should be taken into account. ª 2016 John Wiley & Sons Ltd 369

6 Fig 4. Mild phenotypes. Hands with dyspigmentation and scarring; brown pigmentation with a sharply defined margin in the teeth; feet with speckles and small blisters. Haematological complications are the main predictors of poor prognosis for CEP. Patients who present CEP onset between birth and 5 years of age are more likely to develop severe, progressive haematological complications (Katugampola et al, 2012). The hypothesis that the onset of anaemia, from any cause, could lead to aggravation or initial presentation of the symptoms of CEP and lead to increased production of porphyrins remains unproven, though this theory is worthy of consideration (Fig 7). An exemplar case report showed how a marked worsening of anaemia in a transfusion-dependent patient with bone marrow fibrosis preceded the onset of clinical manifestation of CEP (Verma et al, 2014). The co-inheritance of a thalassaemia trait in CEP patients has also been reported to increase the severity of the phenotype (Maakaron et al, 2012). Thalassaemia might increase the demand for haem products through ineffective erythropoiesis. The anaemia and the accompanying iron overload can induce haem synthesis by increasing ALAS2 activity in consequence of increased translation of ALAS2 mrna. At the same time a deregulation of the ALAS2 gene with a low rate of porphyrin accumulation has been demonstrated in a patient who showed a remarkable improvement in disease manifestations in a setting of iron restriction (Egan et al, 2015). Hypertransfusion in the treatment of severe anaemia may prevent blister formation as the transfused blood suppresses haematopoiesis and the production of endogenous porphyrins (Piomelli et al, 1986; Hogeling et al, 2011). However the secondary iron overload must be taken into account. Other clinical characteristics Erythrodontia and dentine disorders are present in most of CEP patients and are the direct consequences of a diffuse tissue accumulation of porphyrins during teeth development. The teeth, when exposed to long-wavelength ultraviolet light, produce fluorescence due to the porphyrin deposit and also a reddish-brown discoloration even under visible light (Fig 5). The 370 ª 2016 John Wiley & Sons Ltd

7 Fig 5. Moderate Phenotypes. Hands with blisters, ulcers and scarring; erythrodontia; facial dyspigmentation, scabs and scars. porphyrin accumulation can lead to manifestation of symptoms also in cases when a CEP patient is pregnant. A healthy child of a CEP patient showed distal brown pigmentation with a sharply defined margin in the erupted teeth (Hallai et al, 2007). In infants, the calcification occurs before birth during exposure to maternal porphyrin. The toxic effect of sunlight induces alterations of eyelids, conjunctiva and sclera, which represent the underlying pathological mechanism leading to ocular complications in CEP patients. Scarring of eyelids may also result in lagophthalmos with severe keratopathy. Ocular involvement, such as blepharitis, loss of the eyelashes and eyebrows, conjunctivitis, chronic ulcerative keratitis and scleromalacia, is frequent. Bilateral corneal scarring may also occur leading to blindness in most severe cases. Although the ocular damage can be quite serious, it seems that the risk of development of neoplastic conjunctival or corneal cell changes is low (Hillenkamp et al, 2001). Accumulation of uroporphyrin and coproporphyrin was also described in teardrops and a direct correlation between their concentration and severity of ocular involvement was observed (Takamura et al, 2002). Bone development in CEP might result in osteodystrophia combining osteolysis of light exposed extremities and osteoporosis. The bone re-absorption is evident in severe mutilations in terminal digits of the hand while osteoporosis correlates with the severity of anaemia (Katugampola et al, 2012). Reduced bone mineralization and fragility of the long bones and vertebrae leading to fractures and shortened stature can be consequences of the bone marrow expansion, in order to compensate for a severe haemolytic anaemia. It is not clear if there is any impact of serum vitamin D levels on skeletal manifestations, as the majority of the patients also have vitamin D insufficiency or deficiency. Nephrotic syndrome with histological evidence of renal iron deposition has been reported (Lange et al, 1995; Katugampola et al, 2012). Neurological implications are not common in CEP even though CEP cases with neurological manifestation, such as ª 2016 John Wiley & Sons Ltd 371

8 Fig 6. Severe Phenotypes. Extensive facial ulcerations and erosions; distortion of facial features and bilateral significant mutilations of hands as well as shortening and contractures of the digits and limbs. Fig 7. The ALAS2 modifier gene. Anaemia, iron overload and gain of function mutation in the ALAS2 gene enhance functional property of the ALAS2 enzyme. This, in turn coupled with a defective uroporphyrinogen III synthase (UROS), may cause a further increase of URO I and coproporphyrin I accumulation, worsening the CEP phenotype. ALA, Aminolevulinic acid; ALAS2, d-aminolevulinate synthase 2; PBG, porphobilinogen; HMB, hydroxymethylbilane; URO I, uroporphyrinogen I; URO III, uroporphyrinogen III. Parkinson disease and corticobasal syndrome, have been reported (Darwich et al, 2011; Sidorsky et al, 2014). It cannot be excluded that these manifestations could only be age-related, but at the same time, it was presumed that a long-term overproduction of porphyrins and their accumulation in the meninges and brain could contribute to the development of neurodegenerative disorders via tissue damage from porphyrin oxidative stress. Genetic bases Congenital erythropoietic porphyria is characterized by a wide genetic heterogeneity, not only in the type of mutations but also in the gene causing the disease. In the majority of cases, the inheritance of CEP shows a typical recessive pattern, as the disease is not inherited in all generations of all affected families. Until 2007 the marked UROS enzyme deficiency was ascribed only to homozygous or compound heterozygous mutations in the UROS gene [National Center for Biotechnology Information (NCBI) ID 7390]. Recently, three male patients carrying a mutation in the X-linked gene encoding for the transcriptional factor GATA1 (NCBI ID 2623) have been reported to have CEP phenotype (Phillips et al, 2007; Di Pierro et al, 2015). In several CEP cases, the second mutation in the UROS gene was not identified and any acquired UROS or GATA1 mutation was also reported to be present in late-onset cases with a pre-existing myeloid malignancy, suggesting that other genes could be involved in CEP presentation (Sarkany et al, 2011). Phenotypic variability has also been reported in the same family among patients harbouring the same gene defect. The recent description of an ALAS2 gain-of-function mutation as an aggravating factor for the most severe phenotype, underscores the possible implication of modifier genes underlying the CEP phenotype (To-Figueras et al, 2011). A knock-in viable mouse model of human CEP has been generated by gene targeting with murine missense mutations that encode low levels of UROsynthase activity (Bishop et al, 2006; Ged et al, 2006). UROS gene The full-length cdna encoding uroporphyrinogen III synthase was isolated and sequenced in 1988 by screening of a human adult liver cdna library (Tsai et al, 1988). Its length 372 ª 2016 John Wiley & Sons Ltd

9 was 1296 bp with 5 0 and 3 0 untranslated regions of 196 bp and 302 bp, respectively, and encoded a protein of 265 amino acids. Using the cdna as a probe, a single UROS gene was mapped to the telomeric region of the long arm of chromosome 10, more exactly at 10q252 q263 locus (Astrin et al, 1991). The human UROS gene structure is about 34 kb in length and organized in 10 exons that include the untranslated exons 1 and 2A and 9 coding exons from 2B to 10 (Aizencang et al, 2000). The introns show variable length from 02 to89 kb, while exons range from 288 to 423 kb. The gene carries promoters upstream of exon 1 and exon 2, specific for a housekeeping and an erythroid form, respectively. The two forms present a common region containing exons from 2B to 10 and a specific 5 0 UTR exon, while exon 1 is present in the housekeeping form and exon 2A in the erythroid one. The initiation codon is present only in exon 2B, which is common to both forms. Therefore, transcription is regulated by different promoters in a tissue-specific manner, but the resulting enzymes have identical amino acid sequences. Expression arrays revealed that the housekeeping transcript is present in all tissues, while the erythroid transcript occurs only in the erythropoietic tissues. High levels of erythroid expression have been detected in fetal liver, bone marrow and fetal spleen. In contrast, the ubiquitous expression is quite low in most cells with the only exception of the skeletal and heart muscle, caudate nucleus and amygdale, where high expression levels are found. This is consistent with the architecture of the promoter, as it lacks the canonical TATA1 and SP1 sites for initiation of transcriptional activity. On the other hand, the erythroid-specific proximal promoter upstream of exon 2A contains erythroid transcription factor binding sites including GATA1 and NFE2, conferring strong erythroid specificity (Aizencang et al, 2000). The 5 0 end of UROS abuts with the BCCIP gene on the reverse strand in a head to head manner, sharing 277 bp of intergenic region. A functional analysis of this region identified promoter activity in both orientations, thus showing that UROS and BCCIP share a bi-directional promoter for their expression (Meng et al, 2003). UROS mutations Forty-nine different mutations causative for CEP have been reported based on the Human Gene Mutation Database (HGMD, and the literature (Table SII). The majority are point mutations, only six gene rearrangements including long deletions, insertion and del-ins mutations have been described. Twenty-seven mutations are missense mutations, representing the majority of UROS alterations (about 56%). Among the remaining mutations, only one is nonsense, five are splicing defects, six are located in regulatory region, four are deletions, four are insertions and two are del-ins. By employing an E. coli-based expression system, 23 missense mutations were functionally tested by measuring relative enzymatic activities and kinetic stability (Fortian et al, 2009). This experimental evidence suggested that some of the UROS defects alter the catalytic machinery of the enzyme, other mutations impair the stability of the folded conformation, whereas others affect both the protein activity and stability (Table SII). More recent works about the UROS C73R mutation demonstrated that the apparent enzyme activity was reported to decrease over time at a rapid rate due to premature unfolding and quick degradation resulting in undetectable protein levels in the cell (Fortian et al, 2011; ben Bdira et al, 2014). Also the P248Q mutant was associated with a reduced kinetic stability and this could also be valid for the other missense mutations (Blouin et al, 2013). The impairment of stability can be very important, given that UROS, by its nature, is a thermolabile enzyme undergoing slow irreversible denaturation. A subset of mutations (S47P, V99A, A104V and T228M) seems to retain very high synthase activity with no decrease in the stability. S47P is a puzzling mutation, as its homozygous form has been associated with a variable phenotype (Ged et al, 2004; Maakaron et al, 2012). V99A, A104V and T228M have always been found coupled with mutations related to more severe symptoms, suggesting that these polymorphisms might also be present in healthy individuals. Four more missense mutations have recently been identified: G58R (Katugampola et al, 2012), L139K (Guo et al, 2014), P190L (Pandey et al, 2013) and D113V (Di Pierro et al, 2015). All residues except D113 have been reported to be highly conserved among mammals. The G58 residue has been thought to have a role in the catalysis as it is located near the residues that form the surfaces directly above or below the substrate-binding site (Cunha et al, 2008). The substitution of residue L139 by a larger and positively charged arginine was reported to probably cause a change in the packing of the well conserved adjacent residue H140. PolyPhen 20 analysis indicated a deleterious impact on protein function for the P190L mutation and the phenotype associated to this mutation was quite severe. The D113V mutation has been described in double heterozygosis in a GATA1 related case. It has been suggested that this variant could be a polymorphism, because it is also present in a normal population (1000 Genomes Project and ExAc project databases) but this does not exclude its association with a very mild phenotype. Among the five splicing defects, two mutations have been reported to cause an amino acid change (E81D and V82F) even though they produce an mrna lacking exon 4, generating a truncated version of the protein (Xu et al, 1995; Shady et al, 2002). Given this mechanism, both these mutations have been reclassified as splicing defects. Two other mutations (c A>G and c T<G) have been observed far from the splice site consensus sequence, both of which affect branch point of the splicing (Bishop et al, 2010). Other two regulatory mutations (c. 203T>A and c. 219C>G) were described in addition to the four characterized in the erythroid promoter of the UROS gene by Solis ª 2016 John Wiley & Sons Ltd 373

10 et al (2001) and both represented nucleotide substitutions located at the same bases of those previously reported (Table SII). The first one is located exactly at a GATA1 binding site and, similar to c. 203T>C, might have the same impact on impairing GATA1 binding. The second is near a CP2 binding site and similar to c. 219C>A; it is conceivable that this substitution does not alter the essential binding nucleotides (Katugampola et al, 2012). Among the six gene rearrangements only a large deletion of at least 460 bp has been reported to cause the deletion of exon 2 and exon 3 (Katugampola et al, 2012). One deletion (r.148_245del98) and one insertion (r.660_661ins80) might be reclassified as splicing mutations, given that, in the original paper (Boulechfar et al, 1992), the molecular defects were described at the mrna level and were reported to affect exactly the exonic junctions. Nevertheless in this review, they are considered still as a deletion and insertion, as in HGMD. An interesting mutation is the new delins, c.583_589delinsatttttg, located in exon 9 (Katugampola et al, 2012). This mutation deletes bases from 583 to 589, inserting a small nucleotide stretch of the equal length, and causes the substitution of two-residue at position 195 and 197, from Phe to Ile and from Ser to Gly respectively. In particular, S197 has been described as a part of a cluster of residues that presumably represent active site contact regions (Cunha et al, 2008). GATA1 gene The GATA1 gene encodes for a transcription factor that plays a crucial role in the normal development of haematopoietic cell lineages. GATA1 was first identified in 1988 as encoding an erythroid nuclear protein, Eryf1 (Evans et al, 1988). Successively the gene was cloned (Tsai et al, 1989) and assigned to chromosome X by a hybridization technique using a panel of humanrodent DNAs (Zon et al, 1990). In 1991, GATA1 was more precisely located in chromosome Xp1123 (Caiulo et al, 1991). The GATA1 gene encodes a 18 kb mrna yielding two proteins, one full-length 47 kd and one shorter 40 kd protein. The latter, termed as GATA1s, uses an alternative translation initiation site at codon 84, and thus lacks the N-terminal sequence. Structurally, the protein is a single polypeptide presenting two highly conserved zinc finger domains. The C-terminal domain primarily mediates the binding to DNA throughout the sequence (A/T)GATA(A/G) and the N-terminal domain stabilizes DNA interactions by contacting non-canonical GATC and palindromic ATC(A/T) GATA(A/G) motifs in the promoter elements of various target genes including GATA1 itself (Martin & Orkin, 1990; Trainor et al, 1996; Vyas et al, 1999; Newton et al, 2001). The N-terminal finger also binds to co-regulator transcriptional factors, including FOG (Tsang et al, 1997), a member of another zinc finger family that modulates GATA1 activity (Balduini et al, 2004). GATA1 mutations Somatic and germline mutations of GATA1 are related to human pathological phenotypes. Somatic mutations of GATA1 have been described in acute megakaryoblastic leukaemia and transient myeloproliferative disorder in patients with Down syndrome (Gurbuxani et al, 2004). Germline mutations cause haematopoietic disorders, including some forms of thrombocytopenia, with or without thalassaemic feature. Only one mutation (c.646c>t) has been associated with the CEP phenotype of three cases that showed a sexlinked pattern of inheritance, haematological phenotype of b-thalassaemia intermedia and a marked thrombocytopenia in addition to classical clinical symptoms of the disease (Phillips et al, 2007; Di Pierro et al, 2015). Solis et al (2001) reported that two GATA1 binding sites in the erythroid promoter of UROS gene are required for erythroid transcription, demonstrating that mutations that affect the binding of this transcriptional factor markedly impair erythroid-specific transcription. Thus, it is conceivable that mutated GATA1 could also affect the binding to UROS gene promoter causing impairment in gene expression. The reported mutation affects codon 216 and changes arginine to tryptophan (R216W). The same residue is involved in another point substitution described to be responsible for X-linked thalassaemia with thrombocytopenia; in that case, the substitution is from arginine to glutamine (R216Q). The R216 residue is located in a region of the N-terminal zinc finger that is highly conserved. A recent study functionally analysed disease-causing GATA1 mutations (Campbell et al, 2013) that also included R216 mutations. The authors concluded that the R216Q mutation inhibits TAL1 complex recruitment to GATA1 regulated genes rather than impairing the DNA binding, although how R216W damages GATA1 function remains unclear, given that no drastic impact on DNA, FOG1 or TAL1 complex binding was found. It is possible that another cofactor is necessary to bind GATA1 for erythroid transcription of the UROS gene. Allelic frequency (including UROS and GATA1) While considering the allele frequencies of CEP mutations, all the available alleles found in the literature starting from 1997, those described by Desnick and Astrin (2002) and the ones reported by Katugampola et al (2012) (excluding duplicate cases, whenever possible) were taken into account. As in the last comprehensive review (Desnick & Astrin, 2002), we found that among the 291 CEP alleles described, the c.217c>t mutation (p.c73r) is the most common mutation with a frequency of 282%, confirming its role as a mutational hotspot in the UROS gene. The next most common mutations are c.683c>t (p.t228m) and c.743c>a (p.248q) with 55% and 52% frequency respectively, and the next c.139t>c is at an equal level with a frequency of 374 ª 2016 John Wiley & Sons Ltd

11 48%. The c.7g>t (p.l4f), which was among the most common mutations, now has a lower frequency, because only two new alleles have been added to the 2002 collection. It is important to highlight that among the CEP alleles, c.646c>t GATA1 mutation has also been added, but only three alleles have been described so far, confirming rarity of these alleles in CEP patients. It is remarkable that about 76% of CEP alleles remain unknown, indicating that other genes different from UROS or GATA1 could also be involved in the pathogenesis of the disease. Furthermore, the majority of the mutations is pan-ethnic in origin, and have been identified in different racial and demographic groups (Table SI). ALAS2 gene The erythroid ALAS2 gene has been cloned and mapped to the X chromosome in band Xp112 (Bishop et al, 1990; Cox et al, 1990). The gene spans approximately 22 kb and contains 11 exons, with a notable feature, i.e. the presence of a 60-kb intron in the 5 0 -UTR (Conboy et al, 1992). The gene is subject to both transcriptional and post-transcriptional control. In the immediate promoter, several putative erythroid-specific cis-acting elements, including GATA1 and NFE2 binding sites, have been found. An active ironresponsive element (IRE) has been identified in the noncoding exon 1 (Cox et al, 1991). Low iron concentrations promote binding of the IRE binding protein to the ALAS IRE to specifically block its translation, whereas an increase in intracellular iron leads to dissociation of this factor and increase synthesis of ALAS protein. A post-translational control mediated by hypoxia has also been reported (Abu- Farha et al, 2005). ALAS2 is degraded under normoxic condition and repaired for stability and specific activity in oxygen tension reduction (Fig 7). Exon 2 encodes the N- terminal signal sequence required for mitochondrial import, exons 3 and 4 encode a variable portion of the N-terminal end and exons 5-11 the highly conserved C-terminal portion of the mature protein of about 440 amino acids. Two erythroid ALAS mrna transcripts are generated by alternative splicing of exon 4, indicating that there is a structural heterogeneity in the N-terminal region of the protein. This transcript heterogeneity suggests the existence of erythroid ALAS protein isoforms with potentially distinct functional or regulatory roles. ALAS2 mutations A heterogeneous group of point mutations in the catalytic domain of the ALAS2 enzyme has been found to cause X- linked sideroblastic anaemia (Bottomley et al, 1995). In contrast, four gain-of-function mutations resulting in increased ALAS2 activity have been described as being responsible for the X-linked form of Protoporphyria Erythropoietica (Whatley et al, 2008; Balwani et al, 2013; Brancaleoni et al, 2016). Another gain-of-function mutation has been reported in a homozygous UROS patient, who presented a very severe phenotype of CEP. The c.1757a>t substitution causes an amino acid change in the penultimate residue of ALAS2, substituting the well-conserved tyrosine 586 with phenylalanine (To- Figueras et al, 2011). Although the ALAS2 catalytic activity was reported to be marginally increased, especially compared to previously reported gain-of-function of ALAS2 mutations, the authors suggested that this mutation could produce only a mild in vivo effect but, at the same time, this could be sufficient to increase the production of the haem biosynthesis intermediates. Conclusion To date, bone marrow transplantation is the only effective therapy for CEP and has been performed successfully in a small number of CEP patients with severe manifestations (Dupuis-Girod et al, 2005; Faraci et al, 2008). Gene therapy, by transplantation of genetically modified haematopoietic stem cells, has been positively tested in the murine CEP model but has not yet been performed on patients (Robert- Richard et al, 2008). Current efforts to cure the CEP disease are focused on the development of pharmacological approaches as an alternative to bone marrow transplantation. Proteasome inhibitors have been used successfully in a knock-in CEP mouse model to preserve enzymatically active UROS mutants from early degradation. However, efficient and long-term proteasome inhibition is difficult to obtain in vivo, especially in the erythroid lineage. Moreover this treatment cannot constitute a safe therapeutic choice for CEP due to the neurological adverse effects (Blouin et al, 2013). On the contrary, the recent evidence that molecular interactions may partially restore UROS stability and activity throughout the modulation of protein folding pave the way for the design of chaperone-based therapeutic strategies (ben Bdira et al, 2014). The discovery of new pharmacologic ligands able to correct or assist UROS mutant folding, by preventing their recognition by the quality control system, could represent a promising therapeutic approach for CEP. A possible therapeutic role for chronic transfusions and iron chelators in the modulation of the expression of the CEP disease phenotype in vivo is emerging (Egan et al, 2015). However further advances in the understanding of the biochemical mechanism of UROS enzymatic deficiency and elucidation of the molecular pathogenesis are required for new effective therapeutic interventions against CEP. Acknowledgements We thank Dr. Valeria Fiorentino for valuable help in collecting and revisiting CEP cases from the literature. We also thank Hetanshi Naik, Manisha Balwani and Prof. Robert Desnick, Icahn School of Medicine at Mt. Sinai, New York, USA; Zeynep Karakas, Istanbul School of Medicine, Istanbul, Turkey; Paolo Ventura and Prof. Emilio Rocchi, Gruppo Ital- ª 2016 John Wiley & Sons Ltd 375

12 iano Porfirie, Italy for providing patient photos and giving permission to publish them. We thank all of the patients for their collaboration. Dr. De Verneuil H., Inserm, Paris, France; Dr. Pandhi D., Maulana Azad Medical College, New Dehli, India; Dr. C.S. Mugdal, Massachusetts General Hospital, US for their kind help in defining CEP cases. This research was supported in part by grants from the Italian Ministry of Health (RF , RF and GR ) and from Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico. Author contributions Granata graphically designed figures and conducted a detailed literature search to identify all the studies performed from 1997 to date. All authors critically revisited the literature. Supporting Information Additional Supporting Information may be found in the online version of this article: Table SI. Reported cases of CEP from 1997 to Table SII. UROS and GATA1 mutations causing CEP and their allele frequencies. Dr Elena Di Pierro wrote the manuscript, Dr. Valentina Brancaleoni upgraded the genetics section and Dr Francesca References Abu-Farha, M., Niles, J. & Willmore, W.G. (2005) Erythroid-specific 5-aminolevulinate synthase protein is stabilized by low oxygen and proteasomal inhibition. Biochemistry and Cell Biology = Biochimie et Biologie Cellulaire, 83, Aizencang, G., Solis, C., Bishop, D.F., Warner, C. & Desnick, R.J. (2000) Human uroporphyrinogen-iii synthase: genomic organization, alternative promoters, and erythroid-specific expression. Genomics, 70, Arunachalam, M., Bassi, A., Galeone, M., Scarfi, F. & Difonzo, E. (2013) Scleroderma-like hands in a 16-year-old boy. Congenital erythropoetic porphyria (CEP). JAMA Dermatology, 149, Astrin, K.H., Warner, C.A., Yoo, H.W., Goodfellow, P.J., Tsai, S.F. & Desnick, R.J. (1991) Regional assignment of the human uroporphyrinogen III synthase (UROS) gene to chromosome 10q25.2 q26.3. Human Genetics, 87, Balduini, C.L., Pecci, A., Loffredo, G., Izzo, P., Noris, P., Grosso, M., Bergamaschi, G., Rosti, V., Magrini, U., Ceresa, I.F., Conti, V., Poggi, V. & Savoia, A. (2004) Effects of the R216Q mutation of GATA-1 on erythropoiesis and megakaryocytopoiesis. Thrombosis and Haemostasis, 91, Balwani, M., Doheny, D., Bishop, D.F., Nazarenko, I., Yasuda, M., Dailey, H.A., Anderson, K.E., Bissell, D.M., Bloomer, J., Bonkovsky, H.L., Phillips, J.D., Liu, L. & Desnick, R.J. (2013) Loss-of-function ferrochelatase and gain-offunction erythroid-specific 5-aminolevulinate synthase mutations causing erythropoietic protoporphyria and x-linked protoporphyria in North American patients reveal novel mutations and a high prevalence of X-linked protoporphyria. Molecular Medicine (Cambridge, Mass), 19, Baran, M., Eliacik, K., Kurt, I., Kanik, A., Zengin, N. & Bakiler, A.R. (2013) Bullous skin lesions in a jaundiced infant after phototherapy: a case of congenital erythropoietic porphyria. The Turkish Journal of Pediatrics, 55, Bari, A.U. (2007) Congenital erythropoietic porphyria in three siblings. Indian Journal of Dermatology, Venereology and Leprology, 73, Battersby, A.R., Hodgson, G.L., Hunt, E., McDonald, E. & Saunders, J. (1976) Biosynthesis of porphyrins and related macrocycles. Part VI. Nature of the rearrangement process leading to the natural type III porphyrins. Journal of the Chemical Society Perkin Transactions, 1, Battersby, A.R., Fookes, C.J.R., McDonald, E. & Meegan, M.J. (1978) Biosynthesis of type-iii porphyrins: proof of intact ezymic conversion of the head-to-tail bilane into uro gen-iii by intramolecular rearrangement. Journal of the Chemical Society, Chemical Communications, 5, ben Bdira, F., Gonzalez, E., Pluta, P., Lain, A., Sanz-Parra, A., Falcon-Perez, J.M. & Millet, O. (2014) Tuning intracellular homeostasis of human uroporphyrinogen III synthase by enzyme engineering at a single hotspot of congenital erythropoietic porphyria. Human Molecular Genetics, 23, Bensidhoum, M., Larou, M., Lemeur, M., Dierich, A., Costet, P., Raymond, S., Daniel, Y.J., deverneuil, H. & Ged, C. (1998) The disruption of mouse uroporphyrinogen III synthase (uros) gene is fully lethal. Transgenics, 2, Berry, A.A., Desnick, R.J., Astrin, K.H., Shabbeer, J., Lucky, A.W. & Lim, H.W. (2005) Two brothers with mild congenital erythropoietic porphyria due to a novel genotype. Archives of Dermatology, 141, Bhutani, L.K., Sood, S.K., Das, P.K., Mulay, D.N., Kandhari, K.C. & Deshpande, S.G. (1974) Congenital erythropoietic porphyria. An autopsy report. Archives of Dermatology, 110, Bishop, D.F., Henderson, A.S. & Astrin, K.H. (1990) Human delta-aminolevulinate synthase: assignment of the housekeeping gene to 3p21 and the erythroid-specific gene to the X chromosome. Genomics, 7, Bishop, D.F., Johansson, A., Phelps, R., Shady, A.A., Ramirez, M.C., Yasuda, M., Caro, A. & Desnick, R.J. (2006) Uroporphyrinogen III synthase knock-in mice have the human congenital erythropoietic porphyria phenotype, including the characteristic light-induced cutaneous lesions. American Journal of Human Genetics, 78, Bishop, D.F., Schneider-Yin, X., Clavero, S., Yoo, H.W., Minder, E.I. & Desnick, R.J. (2010) Congenital erythropoietic porphyria: a novel uroporphyrinogen III synthase branchpoint mutation reveals underlying wild-type alternatively spliced transcripts. Blood, 115, Blouin, J.M., Duchartre, Y., Costet, P., Lalanne, M., Ged, C., Lain, A., Millet, O., deverneuil, H. & Richard, E. (2013) Therapeutic potential of proteasome inhibitors in congenital erythropoietic porphyria. Proceedings of the National Academy of Sciences of the United States of America, 110, Bottomley, S.S., May, B.K., Cox, T.C., Cotter, P.D. & Bishop, D.F. (1995) Molecular defects of erythroid 5-aminolevulinate synthase in X-linked sideroblastic anemia. Journal of Bioenergetics and Biomembranes, 27, Boulechfar, S., Da Silva, V., Deybach, J.C., Nordmann, Y., Grandchamp, B. & deverneuil, H. (1992) Heterogeneity of mutations in the uroporphyrinogen III synthase gene in congenital erythropoietic porphyria. Human Genetics, 88, Brancaleoni, V., Balwani, M., Granata, F., Graziadei, G., Missineo, P., Fiorentino, V., Fustinoni, S., Cappellini, M.D., Naik, H., Desnick, R.J. & Di Pierro, E. (2016) X-chromosomal inactivation directly influences the phenotypic manifestation of X-linked protoporphyria. Clinical Genetics, 89, Caiulo, A., Nicolis, S., Bianchi, P., Zuffardi, O., Bardoni, B., Maraschio, P., Ottolenghi, S., Camerino, G. & Giglioni, B. (1991) Mapping the gene encoding the human erythroid transcriptional factor NFE1-GF1 to Xp Human Genetics, 86, ª 2016 John Wiley & Sons Ltd