Review: Molecular pathogenesis of hepatic acute porphyrias

Journal of Gastroenterology and Hepatobgy (1996) 11, 1046-1052

MOLECULAR HEPATOLOGY

REVIEW:Molecular pathogenesis of hepatic acute porphyrias BERNARD GRANDCHAMP, HERVE PUY, JEROME LAMORIL, JEAN CHARLES DEYBACH AND YVES N O R D M A N N

INSERM U409, Facultk de Medecine Xavier Bichat, Paris, France

Abstract The molecular cloning of cDNA and genes encoding enzymes of the haem biosynthetic pathway have permitted the genetic defects underlying acute intermittent porphyria (AIP) and hereditary coproporphyria to be unravelled. In AIP, many different gene abnormalities have been documented since 1989. The prevalence of specific defective alleles among AIP families depends on which human population is studied. Founder effects are likely to account for a high frequency of a single mutation in Finland and, to a lesser extent, in Holland, while many other mutations have only been found once, each of them in a single family. In hereditary coproporphyria several different mutations have already been identified since 1994, suggesting that a large allelic heterogeneity also exists. The search for mutations in variegate porphyria has just started since the recent publication of the human cDNA sequence. Direct detection of the mutations using DNA analysis brings a growing contribution to the detection of asymptomatic carriers among relatives of porphyric patients and will, therefore, improve the prevention of acute attacks.

Key words: acute porphyrias, cDNA, coproporphyrinogen oxidase, gene, mutations, porphobilinogen deaminase.

INTRODUCTION Acute intermittent porphyria (AIP), hereditary coproporphyria (HC) and variegate porphyria (VP) are a group of diseases that share many genetic, clinical and biochemical features.' These three porphyrias are transmitted as autosomal dominant disorders with incomplete penetrance; their frequencies range from 1/10000-11100000, AIP being the most common. Clinical manifestations include abdominal pain and neurological dysfunction. These symptoms occur during acute attacks that are often precipitated by drugs, alcohol, low caloric intake or infections; photosensitivity is occasionally present in HC and PV. Biochemical abnormalities are thought to result from primary defects of specilk enzymes along the haem synthesis pathway and consecutive hepatic overexpression of the first enzyme of the pathway, 5-aminoledinate synthase. As a result of these enzymatic disturbances, haem precursors are synthesized in excess in the liver and massive amounts of compounds upstream of the enzymatic block are excreted in the urine and faeces. Since 1972, specific enzymatic defects of porphobilinogen deaminase (PBGD), coproporphyrinogen oxidase (CPO) and protoporphyrinogen oxidase (PPO)

have been identified in AIP, HC and VP, respectively.' During the past decade, the cDNA and, in the two first cases, genes encoding these enzymes have been isolated. These advances, initially achieved for PBGD, have progressively permitted the identification of mutations that account for the corresponding enzymatic deficiencies.

CLONING OF THE cDNA FOR PBGD, CPO AND PPO Porphobilinogen deaminase (EC 4.3.1.8; also referred to as hydroxymethylbilane synthase) is the third enzyme of the haem biosynthetic pathway. It is a cytosolic enzyme that catalyses the stepwise deamination and head-to-tail condensation of four molecules of porphobilinogen (PBG) resulting in the formation of the unstable linear tetrapyrrole 1-hydroxymethylbilane.' The first PBGD probe was a rat cDNA from erythroid tissues.2 This probe was then used for isolating human cDNA from both erythroid and non-erythroid s o ~ r c e s .The ~ ~ ~ primary structure of the erythroid protein deduced from cDNA sequencing consisted of 344 amino acids. Sequencing of the non-erythroid

Correspondence: B Grandchamp, MSERh4 U409, Faculte de Medecine Xavier Bichat, 75018 Paris, France. Accepted for publication 16 April 1996.

Hepatic acute porphyria

cDNA revealed a 5' end totally unrelated to that of the erythroid cDNA with an additional open reading frame in phase with the initiating methionine of the erythroid protein. Thus, it was deduced that the non-erythroid form of PBGD differed from the erythroid enzyme by an additional peptide of 17 amino acid residues at its NH, terminus. Analysis of PBGD mRNA &om different tissues demonstrated that both mRNA species were distributed according to an absolute tissue specificity, the erythroid form being restricted to erythropoietic cell^.^^^ Porphobilinogen deaminase cDNA have been cloned from a variety of organisms from Escherichia coli to human. Comparison of deduced amino acid sequences revealed a high degree of conservation through evolution. This is often useful for the analysis of PBGD mutations, as the replacement of an evolutionary conserved amino, acid in the human protein usually results in an alteration of the enzyme function. Moreover, the E. coli enzyme has been crystallized and its three-dimensional structure has been determined by X-ray crystallography. The strong conservation of structurally and functionally important amino acids allows analysis of the reported mutations for likely effects on the structure and enzymatic properties of the human e n ~ y m e . ~ Coproporphyrinogen oxidase [EC 1.3.3.3.1 is a soluble protein that is localized in the intermembrane space of mitochondria in mammalian cells. It catalyses the sixth step in haem biosynthesis: the conversion of the two propionate groups at positions 2 and 4 of coproporphyrinogen I11 to two vinyl groups, thus producing sequentially harderoporphyrinogen IX and protoporphyrinogen IX.l cDNA probes for murine CPO have been isolated using oligonucleotides synthesized on the basis of peptide sequences inf~rmation.~,~ These probes readily permitted the cloning of human c D N A . ~ Sequencing ,~ of these cDNA revealed the primary structure of the protein. A comparison of the human protein with those from various sources demonstrated that the amino-terminal part of the enzyme is the most divergent, while most of the sequence is highly conserved. Comparison of the amino acid sequence deduced from the nucleotide sequence of the human mRNA with the N-terminal sequence of the mature rat enzyme identifies a long presequence of 110 amino acids.9 This shares some common characteristics with presequences of other proteins situated in the intermembrane space of mitochondria. Portions of the human cDNA encoding the putative mature enzyme have been expressed in E. coli and are active enzymati~ally.~.~ Protoporphyrinogen oxidase [EC 1.3.3.4.1 is the penultimate enzyme involved in the haem biosynthetic pathway. It catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX.' In eukaryotes, PPO is located in the inner membrane of mitochondria. cDNA encoding humanlo PPO has recently been isolated by in vivo complementation of a mutant E. coli strain. The deduced polypeptide consisted of 477 amino acid with a predicted molecular mass of approximately 51 kDa. Although the newly synthesized enzyme must be imported into the mitochondria, no targeting presequence was found.

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ORGANIZATION OF T H E GENES FOR HUMAN P B G D AND CPO The human PBGD gene was cloned and its organization was characterized. It is a single copy gene in the human genome located in the chromosomal region 11q24." It is split into 15 exons spread over 10 kb of DNA (Fig. 1). Two distinct mRNA are produced through alternative splicing of two primary transcripts arising from two promoters. The upstream promoter is active in all cells, the downstream promoter situated at 3 kb of the ubiquitous promoter, is specifically used in developing erythroid cells.I2 The erythroid promoter displays some structural homology with the P-globin gene promoter, including a CAAC motif, two GATA-1 and one NFE-2 binding site. This suggests that some common trans-acting factors coregulate the transcription of these genes during erythroid development.I3 The complete genomic sequence, including the 5' regulatory, 3' untranslated and intronic regions, has recently been published.I4 The human CPO gene has recently been cloned and ~equenced.~ It is a single copy gene that spans approximately 14 kb in the chromosomal region 3qI5 and consists of seven exons and six introns. Multiple transcriptional initiation sites were located using primer extension and RNAse protection assays from various mRNA sources. The results indicate that a single promoter is active but differentially regulated in erythropoietic and non-erythropoietic cells. Computer-assisted analysis of the promoter region revealed a very GC-rich region (77% in a 350 nt region) containing many potential cis-acting regulatory elements. There was no consensus sequence for TATA or CAAT boxes, as is usually observed for promoters of constitutively expressed genes. However, six Sp 1 binding sites were situated upstream of the major initiation sites and two additional recognition sequences were found in exon 1. Furthermore, four GATA sites and the CACCC boxes were found. Spl binding sites are most often present in the promoter region of constitutively or widely expressed genes, while GATA binding sites are commonly found in combination with either CACCC boxes or Spl binding sites in the promoter region of genes specifically expressed or up-regulated in erythroid cells. Indeed, the level of

111 I

5'

1'

101112 131415

/&JG

Erylhoid m R N A

Figure 1 Structure of the PBGD gene.

3'

B Grandchamp et al.

1048 CPO transcripts was found to increase during erythroid differentiation.

MUTATIONS CAUSING AIP The first AIP mutation was reported in a variant form of the disease. Porphobilinogen deaminase is usually decreased in all tissues of patients with AIP. The enzymatic activity is reduced to approximately 50% of enzyme activity in normal subjects and its measurement in erythrocytes is a diagnostic test for the disease. However, in some families, the PBGD defect appears to be restricted to non-erythropoietic tissues.’ A large family from the Netherlands with this variant form of AIP was studied and a mutation G+A was detected in the 5’ splice donor site of intron 1.l6 As exon 1 is absent from the erythroid mRNA, this findmg explained the normal activity of PBGD in the erythrocytes of these patients. Two additional mutations have been found in exon 1 of the gene in different families with the same variant type of AIP.I7J8

In ‘classical’ AIP, approximately 70 different mutations have now been reported (Table 1). No large deletion of the gene has been found and most mutations are single base substitutions. All kinds of mutations have been reported. They result either in the absence of protein encoded by the mutant allele (85% of cases) or in the synthesis of a stable protein with abnormal catalytic properties (approximately 15% of cases). Approximately 20% of mutations were located at exon-intron junctions and prevented the normal splicing of primary transcripts. Another 15% of defects consisted of small insertions or deletions (1-8 nt) in exons and resulted in translational frameshifts leading to premature termination of protein synthesis. In approximately 15% of cases base substitutions created a stop codon leading to incomplete translation of the mRNA. Finally, in approximately 50% of cases, single base substitutions resulted in amino acid substitutions. An instability of the mutant protein was often found, but some amino acid substitutions markedly reduced the catalytic activity without altering the stability of the

Table 1 Reported mutations in the PBGD gene Position

Mutation

Consequences

Reference

Exon 1

3 33

ATG-ATA GCG-GCT

Translation impairment DS

18 17

Intron 1

33+1

gtg-atg

DS

16

Exon 3

76 77

CGC-TGC CGGCAC

R26C R26H

25 26

Exon 4

91 97 I00

A31T

125

GCT-.CACT Del A CAG-rAAG CAG-rTAG ‘ZTG-rTAG

24 25 27 25 19

163 174 182

GCT-TCT Del C Ins G

Frameshift Frameshift

24 24 24

Inrron 5

210+ 1

gta-ata

DS (Del exon 5)

24

Exon 6

218-21 9

Del AG

Frameshift

24

Exon 7

277 293 33 1

V93F K98R G111R

24 25 28

Intron 7

345- 1

cag-caa

AS (Del exon 8)

29

Exon 8

346 347

CGC-TGG CGC-CAG

R116W R116Q

20 30

Exon 9

44 5 446 446 463 470

CGA-TGA CGA-CAA CGA-CTA C AG-TAG Ins A

R149X R149Q R149L Ql55X

Frameshift

25 31 24 32 29

499- 1

cag-caa

AS (Del exon 10)

21

100

Exon 5

Intron 9

GTl-??T AAG-AGG GGA-+AGA

Frameslhift Q34K Q34X L42x A55S

Hepatic acute porphyria

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Table 1 conc.

Position

Mutation

Consequences

Reference

Exon 10

499 500 518 530 593 604 610 612

CGG-TGG CGG-CAG CGGCAG CTG+CGG TGG-TAG Del G CAG-tTAG CAG-tCAT

R167W R167Q R173Q L177R W198X Frameshift Q204X DS (Del9 bp exon 10)

33 27,34 34 27 19 35 30 31

Exon 11

625

GAG-tAAG

E209K

28

Intron 11

652-3

cag+gag

AS (Del exon 12)

33

Exon 12

667 673 673 713 715-716 730-731 734 739 740 742 748 754 755 766 77 1 77 1

GAL-LLU CGA-t GGA CGA-tTGA CTGCGG Del CA Del C T CTT-rCGT TGC-tCGC TGC-TTC Ins 8 bp GAt-tAAA GC C -rACC GCCjGTC CAC-AAC CTG-tCTA CTG+CTC

E223K R225G R225X L238R Frameshift Frameshift L245R C247R C247F Frameshift E250K A252T A252V H2 56N DS (Del exon 12) DS (Del exon 12)

24 25 25 25 19 36 31 36 18 24 24 36 36 27 39 37

Intron 12

771 + 1

gta+ata

DS (Del exon 12)

19

Exon 13

806 820

ACA+ATA GGG-rAGG

T269I G274R

30 30

Exon 14

838 848 886 900

GGA-AGA TGG-tTAG CAG-TAG Del T

G280R W283X Q296X Frameshift

25 30 25 31

Intron 14

912+ 1

gta-ata

DS (Del exon 14)

28

Exon 15

1062 1073

Ins C Del A

Frameshift Frameshift

38 25

~

PBGD, porphobilinogen deaminase; DS, abnormal splicing, mutation in the donor splice site; AS, abnormal splicing, mutation in the acceptor site; Del, deletion; Ins, insertion. Position refers to the numbering of nucleotides in the housekeeping mRNA starting at the initiating codon.

protein. There was no clear relationship between the type of mutation and the severity of clinical symptoms. This is not very surprising as even within a single family the clinical expression of the disease is quite variable. The prevalence of specific AIP mutations is highly variable. While most of the mutations were ‘private’ mutations, each of them being detected only in one family, two mutations are relatively common in restricted geographical areas. In Sweden approximately half the AIP families share the same mutation W198X,I9 while another mutation R116 W20 accounted for only one-third of AIP cases in Holland.

MUTATIONS CAUSING COPROPORPHYRIA Up until the present, seven mutations have been reported in patients with coproporphyria. Two of these mutations are present at the homozygous state in patients with rare variant forms of coproporphyria, while four other mutations have been detected at the heterozygous state in patients with the usual form of the disease. The first mutation to be identified at the CPO locus was found in a patient previously diagnosed as a

B Grandchamp et al.

1050

homozygous case of coproporphyria.21This patient had a severe form of the disease and constantly excreted massive amounts of. coproporphyrin. She was born to first-cousin parents and had a very low residual activity of CPO. Using reverse-transcription, amplification of the cDNA and direct sequencing of the amplified products, we found a point mutation C to T, resulting in an arginine-to-tryptophan substitution.21Sequencing cDNA from both parents demonstrated that they were heterozygotes. Expression of the mutated cDNA in a bacterial system demonstrated that this substitution resulted in the synthesis of an unstable protein with a residual catalytic activity. The finding of an enzyme less stable than normal but still partially active was not surprising in a homozygous patient, as it is likely that a complete defect of CPO activity would not be compatible with life. Another amino acid substitution was found in siblings with a variant form of the disease. Three siblings (two boys, one girl) with intense jaundice and haemolytic anaemia 'at birth, were previously described as having high levels of coproporphyrin in the urine and faeces.22 The pattern of faecal porphyrin excretion was atypical because the major porphyrin was harderoporphyrin. Homozygosity was suggested by the fact that the level of lymphocyte coproporphyrinogen III oxidase was 10% of controls in the siblings and 50% of normal in both parents (who showed only mild abnormalities of porphyrin excretion). cDNA sequencing revealed an A to G transition that led to the replacement of a lysine residue by glutamic acid at position 404 (K404E) in the mutated protein.23Expression of the protein encoded by the mutant cDNA was obtained in E. wli, and enzymatic studies showed a decreased aflinity of the abnormal enzyme for its substrates; this accounted for the accumulation of coproporphyrin and harderoporphyrin. In patients with the more usual heterozygous form of coproporphyria, the causal mutations have been identified in four cases. These were detected by sequencing the exons of the CPO gene after PCR amplification. A summary of the gene abnormalities found in coproporphyria is shown in Table 2.

IMPLICATIONS OF MOLECULAR DIAGNOSIS OF HEPATIC ACUTE PORPHYRIAS While most carriers of the gene defect remain asymptomatic, attacks of acute porphyria in AIP, H C Table 2 Reported mutations in the CPO gene

Position Exon 1 Exon 1 Exon2 Exon4 Exon 5 Exon6 Intron 6

129 484 545

883 991' 1210* 1277

Mutation

Consequences

Ins 5 bp Frameshift Del 21 bp Del7 aa G-A G189S C-G H295D C-T R331W K404E A-*C G-A DS (Delexon6)

Reference 40

40 41

40 21

23 10

CPO, coproporphyrinogen oxidase. Mutations found in patients with homozygous forms of the disease.

and VP are often precipitated by environmental factors. In addition, the severity of attacks is often determined by inappropriate drug prescriptions. For these reasons, the early detection of gene carriers is important in the prevention of acute attacks because affected patients can be advised to avoid precipitating factors.' Until the recent identification of causing mutations, the diagnosis of gene carriers among relatives of porphyric patients was based upon the detection of specific enzymatic defects. Enzyme assays were usually performed on erythrocytes in AIP and on lymphocytes or fibroblasts in H P and VP. However, in all three porphyrias there was some overlap between enzymatic activities in normal controls and patients and the results of tests were not always conclusive. It follows, therefore, that DNA analysis may provide a more reliable approach to diagnosis. In families with a known mutation, the detection of asymptomatic carriers is quite simple in a molecular genetics laboratory using one of the numerous techniques available based on the in vitro amplification of a fragment of DNA that may contain a specific mutation. In AIP and H C there is a high degree of allelic heterogeneity and, consequently, the main difficulty is to initially determine the mutations in newly diagnosed families. In geographical areas where a specific mutation has a high prevalence, it would seem logical to search for this particular defect. In the majority of cases, however, the most appropriate strategy is to study the exons of the PBGD or CPO gene in AIP and HC, respectively. This can be achieved through the sequencing of an amplified DNA fragment either directly or after these fragments have been screened for the presence of mutations using techniques such as denaturing gradient gel electroph~resis~~ or single strand conformational .polymorphism.25 In summary, there is little doubt that DNA analysis will be the method of choice to detect presymptomatic carriers and, consequently, to improve the prevention of acute attacks in these porphyrias.

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20 Gu XF, De Rooij JS, Lee F et al. High prevalence of a point mutation in the porphobilinogen deaminase gene in Dutch patients with acute intermittent porphyria. Hum. Genet. 1993;91: 128-30. 21 Martasek P, Nordmann Y, Grandchamp B. Homozygous hereditary coproporphyria caused by an arginine to tryptophane substitution in coproporphyrinogen oxidase and common intragenic polymorphisms. Hum. Mol. Genet. 1994;3: 477-80. 22 Nordmann Y, Grandchamp B, De Verneuil H, Phung L, Cartigny B, Fontaine G. Harderoporphyria: A variant hereditary coproporphyria. 3 Clin. Invest. 1983; 72: 113949. 23 Lamoril J, Martasek P, Deybach JC, Da Silva V, Grandchamp B, Nordmann Y. A molecular defect in coproporphyrinogen oxidase gene causing harderoporphyria, a variant form of hereditary coproporphyria. Hum. Mol. Genet. 1995;4: 275-8. 24 Gu XF, De Rooij F, Voortman G et al. Detection of eleven mutations causing acute intermittent porphyria using denaturing gradient gel electrophoresis. Hum. Genet. 1994; 93: 47-52. 25 Kauppinen R, Mustajoki H, Pihlaja H, Peltonen L, Mustajoki P. Acute intermittent porphyria in Finland: 19 mutations in the porphobilinogen deaminase gene. Hum. Mol. Genet. 1995; 4: 215-22. 26 Llewellyn DH, Whatley S, Elder GH. Acute intermittent porphyria caused by an arginine to histidine substitution (R26H)in the cofactor-binding cleft of porphobilinogen deaminase. Hum. Mol. Genet. 1993; 2: 1315-16. 27 Mgone CS, Lanyon WG, Moore MR, Connor JM. Detection of seven point mutations in the porphobilinogen deaminase gene in patients with acute intermittent porphyria, by direct sequencing of in vitro amplified cDNA. Hum. Gener. 1992;90: 12-16. 28 Gu XF, De Rooij F, De Baar E et al. Two novel mutations of the porphobilinogen deaminase gene in acute intermittent porphyria. Hum. Mol. Genet. 1993; 2: 1735-6. 29 Schreiber WE,Rozon C, Fong F, Jamani A. Detection of polymorphisms and mutations in the porphobilinogen deaminase gene by nonisotopic SSCP. Clin. Chem. 1994; 40: 1982-3 [Letter]. 30 Mgone CS, Lanyon WG, Moore MR, Louie GV, Connor JM. Identification of five novel mutations in the porphobilinogen deaminase gene. Hum. Mol. Genet. 1994;3: 809-1 1. 31 Delfau MH, Picat C, De Rooij F et al. Molecular heterogeneity of acute intermittent porphyria: Identification of four additional mutations resulting in the CRIMnegative subtype of the disease. Am. J. Hum. Genet. 1991;49: 421-8. 32 Scobie GA, Llewellyn DH, Urquhart AJ et al. Acute intermittent porphyria caused by a C-T mutation that produces a stop codon in the porphobilinogen deaminase gene. Hum. Genet. 1990; 85: 6314. 33 Gu XF,De Rooij F, Voortman G, Te Velde K, Nordmann Y, Grandchamp B. High frequency of mutations in exon 10 of the porphobilinogen deaminase gene in patients with a CRIM-positive subtype of acute intermittent porphyria. Am. J. Hum. Genet. 1992; 51: 660-5. 34 Delfau MH,Picat C, De Rooij F W et al. Two different point G to A mutations in exon 10 of the

1052 porphobilinogen deaminase gene are responsible for acute intermittent porphyria. 3. Clin. Invest. 1990; 86: 151 1-16. 35 Schreiber WE, Fong F, Jamani A. Frameshift mutations in exons 9 and 10 of the porphobilinogen deaminase

gene produce a crossreacting immunological material (GRIM)-negative form of acute intermittent porphyria. Hum. Genet. 1994; 93: 552-6. 36 Mgone CS, Lanyon WG, Moore MR, Louie GV, Connor JM. Detection of a hgh mutation frequency in exon 12 of the porphobilinogen deaminase gene in patients with acute intermittent porphyria. Hum. Genet. 1993; 92: 619-22. 37 Daimon M, Yamatani K, Igarashi M et al. Acute

intermittent porphyria caused by aG to C mutation in exon 12 of the porphobilinogen deaminase gene that results in exon skipping. Hum. Genet. 1993; 92: 549-53.

B Grandchamp et al. 38 Daimon M, Yamatani K, Igarashi M et al. Acute

intermittent porphyria caused by a single base insertion of C in exon 15 of the porphobilinogen deaminase gene that results in a frame shift and premature stopping of translation. Hum. Genet. 1994; 93: 533-7. 39 Grandchamp B, Picat C, De Rooij F et al. A point mutation G+A in exon 12 of the porphobilinogen deaminase gene results in exon skipping and is responsible for acute intermittent porphyria. Nucleic Acids Res. 1989; 17: 6637-9. 40 Lanoril J, Deybach JC, Puy H, Grandchamp B,

Nordmann Y. Three novel mutations in the coproporphyrinogen oxidase gene. Hum. Mutation 1996 (in press). 41 Fujita H, Kondo M, Taketani S ef al. Characterization and expression of cDNA encoding coproporphyrinogen oxidase from a patient with hereditary coproporphyria. Hum. Mol. Genet. 1994; 3: 1807-10.