Variable phenotypic expression of genotypic abnormalities in the porphyrias

Clinica Chimica Acta. 217 (1993) 29-38 © 1993 Elsevier Science Publishers B.V. All rights reserved. 0009-8981/93/$06.00

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CCA05581

Variable phenotypic expression of genotypic abnormalities in the .porphyrias J.T. H i n d m a r s h Departments of Pathology and Biochemistry, Universityof Ottawa and Ottawa General Hospital, Ottawa (Canada) (Received 22 June 1992; revision received 31 August 1992; accepted 1 September 1992) Key words: Porphyria; Acute intermittent porphyria; Porphyria variegata; Hereditary coproporphyria; Porphobilinogen deaminase; Heine synthesis; Alcohol; Drugs; Estrogen; Progesterone; Gene; Chromosome; mRNA; Mutation

Summary The inherited porphyrias are the consequence of inherited deficiencies of enzymes in the heine synthesis pathway; they exhibit classical Mendelian inheritance patterns. The acute porphyrias (acute intermittent, porphyria variegata, hereditary coproporphyria) result from 50% (approx.) deficiencies of specific enzymes, which demonstrate autosomal dominant inheritance. However, only approx. 10% of subjects who inherit a porphyrin enzyme deficiency develop the corresponding acute porphyria and in most instances there is no obvious reason why one patient with an enzyme deficiency is symptomatic whereas enother is not. Control of heme synthesis is achieved by the represser effect of heme on the enzyme ALA synthase. Acute attacks of porphyria can be precipitated in susceptible persons by drugs, ethanol, starvation, hormones, stress and infection. The mechanism is usually by induction of ALA synthase activity. The molecular biology of porphyria variegata and hereditary coproporphyria is largely unexplored. Acute intermittent porphyria is due to a partial deficiency of the enzyme porphobilinogen deaminase in the liver. The location of the gene for this enzyme has been identified on the long arm of chromosome I 1. Acute intermittent porphyria is a genetically heterogenous disease with the abnormality frequently being a point mutation affecting synthesis of the enzyme,

Correspondence to: Dr. J.T. Hindmarsh, Section of Biochemistry,Ottawa General Hospital, 501 Smyth Road, Ottawa, Ontario, KIH 8L6, Canada.

3o Introduction The porphyrias are a group of Jise:ases with diverse clinical features which are the consequence of inherited and acquired disorders of heme synthesis. Whereas most exhibit a consistent Mendelian inheritance pattern for their enzyme abnormalities, mahy s u b ~ t s with an enzymatic defect do not suffer the clinical features of the disease. Consequently, ~emonstration of a genetic defect and/or an associated enzyme deficiency is not sufficient to diagnose clinically overt porphyria and the older techniques of demonstrating suhstrate accumulation in various body fluids remain the mainstay of biochemical diagnosis. This paper will review which factors are influential izi producing clinical expression of the acute porphyrias and the current status of genetic research in these diseases. Factors which Determine whether an Acute Porphyria will he Clinically Expressed

A classification of the porphyrias is shown in Table I which also records the extent of enzyme deficiencies encountered in patients with specific porphyrias. Those porphyrias where enzyme activity is particularly low (homozygous porphyr~as: congenital erythropoietic porphyria, hepatoerythropoietic porphyria) are clinically severe and most patients who inherit the responsible enzyme defi,',ency are afflicted with the clinical disease, often from early childhood. On the other hand, in patients with the heterozygous (autosomal dominant) porphyrias such as acute intermittent, porphyria variegata and hereditary coproporphyria, who retain 50% activity of the deficient enzyme, heme synthesis can often proceed unimpaired unless stressed by an increased requirement for heme. The synthetic pathway f,:r heme is shown in Fig. I. in the liver, flow through the pathway is controlled by heine inhit~ition of ALA synthase. Heme appears to achieve this control largely by inhibition of the synthesis of ALA synthase at the transcriptional and translational levels, but also by inhibition of enzyme transfer from the cytosol into the mitochondria [!]. These effects are apparently reversed if the intracellular free heine pool is deficient and this results in a substantial increase in enzyme activity with accumulation of excess metabolites proximal to the enzymatic block. Acute intermittent porphyria is the most studied of the acute porphyrias. Subjects with porphyria variegata and hereditary coproporphyria may have similar acute (neurovisceral) features and the factors which tend to precipitate acute attacks in acute intermittent porphyria commonly also do so in variegata and hereditary coproporphyria; the following discussion can thus be considered to apply to all three dPeases, Factors which may precipitate aa acute attack of porphyria in susceptible individuals are listed in Table [!; these have recently been re,viewed by Kauppinen and Mustajoki [2]. However, in many instances, there is no obvious reason why acute attacks occur and why the disease is clinically expressed in some patients but not in others with the same inherited porphyrin enzyme deficiency, Only approx. 10% of subjects with documented deficiencies of porphobilinegen deaminase become symptomatic, A typical family investigated by Kappas et al. [i] had ]7 members with documented deficiencies of porphobilinogen deaminase; only 2 subjects, however, had increased urinary excretion of porphobilinogen and over a period of IS years only 1 of these developed frank clinical acute intermittent porphyria.

Mode of inlk~-itanee

Autosomal dominant

Liver

Uroporphyrinogen decarboxylase ~P/~ liver-sporadic form; 50% erythrocytes familial form, segregation not exact, see Ref. 53) Uroporphyrinogen deearboxylase fvariable) Uroporphyrinogen deearboxylase (25% erythorcytes, fibroblasts) Coproporphyrinogen oxidase (100/o,lymphocytes) Ferrochelatase (< 30%, fibroblasts, lymphocytes)

Liver

Liver

Liver

ALA dehydratase (por~hobilinogen synthase) (< ! 2% erytbrocytes) Protoporphyrinogen oxidase (50%. fibroblasts, lymphocytes) Coproporphyrinogen oxidase (50%. fibroblasts, lymphocytes)

Liver

Porphobilinogen deaminase (50%. erythrocytes)

Liver Liver (and erythropoietic cells) ? Erythropoietic cells (and liver?)

Erythropoietic cells

Predominant site(s) of metabolic expression

UroporphyrinogenAll synthase (15%, erythrocytes)

Enzyme defect (approx. percentage of normal activity and tissue assayed; within each disease, enzyme, activity may vary in differc~,t pedigrees)

aHepatoerythropoietic porphyria is a homozygous form of familial porphyria cutanea tarda: homozygous forms of acute intermittent porphyria, porphyria variegata and hereditary coproporphyria have also been described [!,44!. Enzymatic data taken from references [!,45-52].

Hereditary coproporphyria porphyria

Porphyrius producing both acute and cutaneous manifestations Porphyria variegata Autosomal recessive

Acute porphyrias Porphyrias producing acute manifestations only Acute intermittent Autosomal dominant porphyria ALA dehydratase Autosomal recessive deficiency

Non-acute porphyrias Porphyrias producing cutaneous lesions Autosomal recessive Congenital erythropoietic porphyria Two types: sporadic a.~d Porphyria cutanea tarda famifial, autosomal dominant Acquired Toxic porphyria Autosomal recessive Hepato-erythropoietic porphyriaa Autosomal recessive Harderoporphyria Autosomal dominant Erythropoietic protoporphyria

Disease

Classification of the porphyrias

TABLE I

32

GLYCINE+ SUCCXNYLCOA

! Pyr|doxalPhosphate /

Synthase

AHINOLEVULINICACTD

Negattve Fe~dback Znh|bitten

!

2

I

Porphob411hogan9eamlnase

HYDROXYNETHYLBXLANE

J i~)Rr~~

3

ynthase

Uro~.~or~phyr 1hoganOecarboxylllm CO~PHYRZNOOENXI| Coprop~rphyrtnogan Ox1dane

~Spontaneous UROPORPI4YRXNOOEN |

4

~lroporphyrtnogen Oeco~rboxylase

COPROPORPH~RXHOOEN | 5

14ARIIER~P~Jq|NGGI~N Cc~ro~rrphyr t~gen Oxtdsse

6

Protop~rphyrt nogefi Oxtdale

?

PROTOP~PlWR][N

Farroc~olatsu

8

Fig. I. The pathway of heine synthesis inc~,ding sites el'enzyme insulliciency in the porphyries. I. ALA 4eh:)drata~e deficiency, tyrosinemia, lead poisonins. 2. Acute intermittent porphyria. 3. Congenital erythropoietic porphyris. 4. Po~hyria cutan~.a tardy, hepatoerythropoieti¢ porphyria. Ioxic porphyria -commonest site. 5. Hereditary ¢oproporphyria, lead p~isoning. 6. Harderophorphyria. 7. Porphyria variegate. 8. ~.rythropoietic protoporphyri~, lead poisoning.

33 TABLE 11 Factors which precipitate acute attacks in patients with acute porphyrias Drugs Ethanol Starvation Hormones Stress Infection

The best known precipitants of acute attacks in the acute porphyrias are barbiturates and sulfonamides. Wadenstrom [3] recalls that patients with attacks of acute intermittent porphyria from Northern Sweden often did better in the old days when, during an attack they simply retired to their huts with a bowl of porridge, than those who were transported by air to hospital and exposed to modern drug therapy. Also, Dean [4] states that porphyria variegata was largely unknown in South Africa until the advent of barbiturates and sulfonamides. The mechanism of porphyrinogenicityinduced by drugs is multifaceted and complex. Many drugs are known to produce this effect and the mechanism in many is thought to be induction of hepatic cytochrome P-450 with consequent increase in heme demand leading to induction of hepatic ALA synthase [5]. Phenobarbitone induces increased synthesis of the apoprotein of cytochrome P-450 [6] whereas 2-aUyl-2-isopropylacetamide (AIA), a structural analog of Sedormid, destroys cytochrome P-450 heine [7]. Griseofulvin and 3,5.diethyoxycarbonyl-l,4-dihydrocollidine (DDC) destroy heme and inhibit ferrochelatase [8]; heme is converted to N-alkyl porphyrins which inhibit ferrochelatase. With many other drugs, however, the mechanisms are less clear but the common factor appears to be a compensatory increase in ALA synthase activity. Alcohol (ethanol) may precipitate acute attacks by increasing heme utilization and blocking its synthesis [9]. Ethanol is also reported to inhibit ALA dehydratase activity in vivo [10]. It is chtimed that acute porphyria can be induced in normal persons as well as in porphyrics by carbamazepine therapy probably as a consequence of inhibition of porphobilinogen deaminase by the drug [1 I]. Sulfonamides may also exert their effect by inhibiting the action of hepatic porphobilinogen deaminase [12]. Starvation may precipitate acute attacks in porphyrics and c,rbohydrate administration can ameliorate them even when the diet is adequate. These effects are accompanied by coincident appropriate changes in urine porphobilinogen excretion in patients with acute intermittent porphyria [13]. In rat liver, AIA induction of ALA synthase is inhibited by glucose administration in vivo and in vitro [14,15]. Sex hormones clearly play a major part in determining the course of acute porphyrias. Acute attacks are rare (but not unknown) before puberty. Also symptomatic cases are more common in women and the clinical features may be exacerbated just prior to menstruation [16]. Estrogen and progesterone administration aggravate acute porphyrias [17,18] probably by inducing the formation of cytochrome P-450 in the liver [19].

34

There is evidence that patients who experience acute attacks of acute intermittent porphyria (and porphyria variegata) may have impairment of 5-alpha reduction of steroid hormones to their corresponding metabolites in the liver, whereas latent cases (those who have inherited a porphyrin enzyme deficiency but who do not experience acute attacks) exhibit normal steroid reduction [20]. This may be a factor in determining the clinical expression of the disease as hepatic ,~ LA synthase in chick embryo hepatocytes is particularly well induced by steroids with a 5-beta configuration [1]. It remains to be seen, however, whether this is a common phenomenon. There is no clear evidence that impaired 5-~lpha steroid reduction is inherited and it may be an acquired phenomenon [20]; also phenobarbitone impairs 5-alpha reductase activity [21]. Stress and infection induce acute attacks in porphyrics [5] but the causes have not been clearly determined. The same factors responsible for precipitating acute attacks in acute intermittent porphyria often also do so in porphyria variegata and hereditary coproporphyria. Clinical expression of these diseases is also very variable. One of the better markers for these diseases is elevated stool porphyr~ns (protoporphyrin in variegata, coproporphyrin in hereditary coproporphyria). Patients who have once been symptomatic usually demonstrate this abnormality even when they are in an asymptomatic phase. However, patients who never had symptomatic disease commonly have normal stool porphyrin levels. In summary, there is no obvious genetic explanation to date as to why some patients who inherit porphyrin enzyme deficiencies develop symptomatic disease whereas others do not. Even if 5-alpha reductase deficiency is a factor in producing clinical expression, it may well be an acquired rather than an inherited phenomenon.

Molecular Biology of the Acute Porphyrias The molecular biology of the acute porphyrias has recently been reviewed by Nordmann et al. [22]. The molecular biology of porphyria variegata and hereditary coproporphyria is largely unexplored except for a report of close linkage between tl~e genes for porphyria variegata and alpha-I antitrypsin on chromosome 14 [23]. Most molecular biology research has focused upon acute intermittent porphyria, the commonest of the acute porphyrias. Two isoenzymes of porphobilinogen deaminase have been identified in humans, one from erythrocytes, another from non-erythropoietic cells. These differ in their amino terminus, the latter containing 17 more amino acids than the former [24]. These isoenzymes are generated by the use of different promoters and differential splicing from a single gene of 10 kb on the long arm of chromosome i ! [2,25,26]. Most cases of acute intermittent porphyria demonstrate reduced erythrocyte porphobilinogen deaminase activity. However rare cases demonstrate normal erythrccyte activity, the deficiency presumably bein~gconfined to non-erythropoietic tissues [27]. This latter variety of acute intermittent porphyria can result from a single base substitution (guanine to adenine) in the 5' splice donor site of intron I of the porphobilinogen deaminase gene [28]. This causes failure of normal expression of mRNA for the non-erythropoietic enzyme while sparing the shorter erythrocyte

35 enzyme. It can also result from a guanine to thymidine substitution in the same region [29]. Also, based upon a comparison of erythrocyte porphobilinogen deaminase activity and measurement of its mass by immunological methods, two varieties of acute intermittent porphyria with reduced erythrocyte enzyme activity have been reported [30]. These can be defined as cross-reactive immunological material (CRIM) negative and CRIM-positive. In the former (the common type, 85% of cases) the activity measurement of porphobilinogen deaminase in erythrocytes corresponds to its mass measurement. In the latter, of which there are two main types, the mass measurement of enzyme protein exceeds that of the activity by various amounts indicating the presence of an enzyme protein which is functionally abnormal. CRIM-positive AIP has been shown to be associated with point mutations (base substitutions, guanine to adenine) mainly at exon 10 in the porphobilinogen deaminase gene which result in production of aberrant forms of mRNA which encode enzyme proteins with abnormal structure and impaired catalytic activity that are nevertheless irr.munologically potent [31,32]. Of the common (CRIM-negative) variety of AIP, one case has been shown to result from a cytosine to thymidine mutation in one allele of the porphobilinogen deaminase gene which converts a codon for giutamine to a stop codon [33]. Also, screening of 33 Swedish families with acute intermittent porphyria showed that 15 had a mutation (CRIM-negative) in which a base substitution (guanine to adenine) in exon 10 of the porphobilinogen deaminase gene changes the codon for tryptophan 198 to a stop codon [34]. A further case of CRIM-negative acute intermittent porphyria has been ascribed to a deletion of approx. 50 bases from porphobilinogen deaminase mRNA [35] due to an abnormality at exon 13 (G.H. Elder, unpublished results) and other point mutations producing CRIM-negative disease have been described [36]. It is evident that acute intermittent porphyria is a genetically heterogenous disease [36] and that different point mutations are often the cause. Polymorphisms linked to the porphobUinogen deaminase locus may assist in determining inheritance patterns of the disease at least in some families [37-42].

Molecular Diagnosis of the Porphyrlas Although genetic analysis may eventually make possible the prenatal diagnosis of all the porphyrias, because of the small likelihood of clinical expression of the heterozygous acute porphyrias (10% of patients with a partial deficiency of the respective heme synthesis enzyme) molecular diagnosis is a poor indicator of whether a patient will develop frank disease and this probably should not be used as an indication for termination of pregnancy. At the present time there is no evidence that any one of the many genetic abnormalities that produce acute porphyria is more likely to produce clinical expression than any other and, therefore, identification of the genetic abnormality adds little to clinical management of the patient. With the autosomal recessive (severe) porphyrias (such as congenital erythropoietic porphyria and hepatoerythropoietic porphyria) where the likelihood of

36 clinical expression is high, prenatal diagnosis might be useful and has been employed as an indication for pregnancy termination in the former disease [43]. Conclusion Acute attacks o f porphyria may be precipitated in susceptible persons by many factors and it is important that these be avoided. While identification o f the molecular genetic abnormalities have greatly added to the understanding o f the porphyrias, because o f the variability of clinical expression in the he~erozygous forms, at the present time, clinical diagnosis must still rely upon clinical expression of the disease associated with biochemical identification o f accumulation o f intermediary metabolites o f the heme synthesis pathway. Aelmowledgment Thanks are due to Professor G.H. Elder for advice and help with this manuscript. References I

2 3 4 5 6 7

8 9 10 I! 12 13 14

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38 36 Delfau MH, Picat C, DeRooij F et al Molecular heterogeneity of acute intermittent porphyria: identification of four additional mutations resulting in the CRIM negative subtype of the disease. Am J Hum C,enet 1991;49:421-428. 3"/ LlewellynDH. Kalsheker NA, Harrison PR et al. DNA polymorphism of human porphobilinogen deaminase gene in acute intermittent porphyria. Lancet 1987;2:706-708. 38 Lee JS, Anvret M, Lindsten J e t al. DNA polymorphisms within the porphobilinogen deaminase gene in t;~o Swedish families with acute intermittent porphyria. Hum Genet 1988;79:379-381. 39 Lee JS, Lindsten J, Anvret M. Haplotyping of the human porphobilinogen deaminase gene in acute intermittent porphyria by polymerase chain reaction. Hum Genet 1990;84:241-243. 40 Scobie GA, Urquhart AJ, Elder GH et al. Linkage disequilibrium between DNA polymorphisms within the porphobilinogen deaminase gene. Hum Genet 1990;85:157-159. 41 gauppinen R, Peltonen L, Palotie A, Mustajoki P. RFLP analysis of three different types of acute intermittent po~hyria. Hum Genet 1990;85:!60-164. 42 Lee JS. Lundin G, Lannfelt L et al. Genetic heterogeneity of the porphobilinogen deaminase gene in Swedish families with acute intermittent porphyria. Hum Genet 1991;87:484-488. 43 Kaiser IH. Brown amniotic fluid in congenital erythropoietic porphyria. Obstet Gynecol 1980;56:383-384. 44 BeukeveldGJ, Wolthers BG, Nordmann Y. Deybach JC, Grandchamp B, Wadman SK. A retrospective study of a patient with homozygous form of acute intermittent porphyria. J Inherit Metab Dis 1990:13:673-683. 45 Bonkowsky HL. Bloomer JR, Ebert IS. Mahoney MJ. Heine synthetase deficiency in human protoporphyria. J Clin Invest 1975;56: I 139- 1148. 46 de Verneuil H, Aitken G. Nordmann Y. Familial and sporadic porphyria cutanea, two different diseases. Hum Genet 1978;44:145-151. 47 Elder GH, Smith SG, Herraro C et al. Hepatoerythropoietic porphyria, a new uroporphyrinogen decarboxylase defect or homozygous porphyria cutanea tarda. Lancet 1981;1:916-919. 48 Deybach JC, de Verneuil H, Nordmann Y. The inherited enzyme defect in porphyria variegata. Hum Genet 1981:58:425-428, 49 Doss M, Schneider J, yon Tiepermann R, Brandt A. New type of acute porphyria with porphobilinogen synthase (delta aminolevulinic acid dehydratase) defect in the homozygous state. Clin Biochem 1982;15:5:?-55. 50 Nordmann Y, Grandchamp B, de Verneuil K, Phung L, Cartigny B, Fontaine G. Harderoporphyria, a variant hereditary coproporphyria. J Clin Invest 1983:72:1139-1149, 51 Koszo F, Eider GH, Roberts A, Simon N, Uroporphyrinogen decarhoxylase deficiency in

hepatoerythropoietic porphyria: further evidence for genetic heterogeneity, Br J Dermatol 1990;122:365-370. 52 PlewinskaM, Thunell S, Holmberg L. Wetmur JG, Desnick RJ. Delta-aminolevulinate dehydratase deficient porphyria: identification of the molecular lesions in a severely affected homozygote. Am J Hum Genet 1991:49:167-174. 53 Elder GH, Roberts AG, de Salamanca RE. Genetics and pathogenesis of human uroporphyrinogen decarboxylase defects. Clin Biochem 1989;22:163-168.