J. Clin Pathol 1993;46:977-981 977

;; Leaders :;:::::;::;::;::;::;::;::;::;: ;:; ;;;;;;;;;;:;; J Clin Pathol: first published as 10.1136/jcp.46.11.977 on 1 November 1993. Downloaded from Molecular genetics of disorders of haem biosynthesis

G H Elder

Introduction chelatase,20 and ,B globin ,2' contains Haem is synthesised in all animal cells from sequences that interact with the trans-acting succinyl CoA and glycine by a sequence of erythroid specific factors, GATA-1 and NF- reactions catalysed by eight enzymes.1 E2, and regulate transcription during ery- Absence of any one of these enzymes is throid differentiation.5 The 5' untranslated incompatible with life because haem is an region (UTR) of ALAS2 mRNA, unlike essential component of respiratory . ALAS 1 mRNA, contains a sequence struc- Partial deficiencies occur, however, and cause ture motif that closely resembles the iron disease (table 1).12 The activity of 5-amino- responsive elements (IREs) found in the 5' laevulinate (ALA) synthase, the first and rate UTR of ferritin mRNA and the 3' UTR of controlling enzyme of the pathway, is transferrin receptor mRNA.i22 Iron deficiency decreased in the bone marrow in hereditary leads to high affinity binding of an IRE-bind- sideroblastic anaemias,3 while deficiency of ing to these elements with consequent each of the subsequent enzymes produces a inhibition of translation of ALAS2 and fer- particular type of (table 1) .1 2 The ritin mRNAs and stabilisation of transferrin enzyme defect in each porphyria is inherited receptor mRNA,22 a mechanism that allows (table 1), except in type I porphyria cutanea co-ordination of protoporphyrin formation tarda which appears to be an acquired dis- with iron supply. The presequences of both order. The clinical features and laboratory ALAS isoenzymes also contain a conserved, diagnosis of the have been haem regulatory motif which is involved in reviewed.' 2 inhibition by haem of their transport into Genes or cDNAs have now been isolated mitochondria.424 and characterised for all but two of the The discovery that ALAS2 maps to the X human enzymes (table 2). These advances prompted a search for muta- have been followed rapidly by identification tions in this in sideroblastic anaemia. A of mutations in the corresponding diseases T-÷A transition that converts isoleucine to

and by the application of DNA techniques to asparagine has recently been identified in a http://jcp.bmj.com/ the management of families with these condi- man with severe, pyridoxine responsive tions. This review summarises current infor- sideroblastic anaemia.25 This mutation is in a mation about the molecular genetics of each highly conserved region of exon 9 that is close disorder. to the putative pyridoxal phosphate binding lysine residue and thus may impair binding of Hereditary sideroblastic anaemias the cofactor. This man had no family history

The hereditary sideroblastic anaemias are a of anaemia but a second mutation in the on September 27, 2021 by guest. Protected copyright. mixed group of disorders in which the most ALAS2 gene has now been found in a family frequent pattern of inheritance indicates link- with X linked pyridoxine responsive sidero- age to the . Bone marrow blastic anaemia.26 Because the low level of ALA synthase activity is usually decreased.' expression of ALAS 1 in erythroid cells is Pyridoxal phosphate is an essential cofactor unlikely to be sufficient by itself for erythro- for this enzyme and patients may respond to poiesis,5 other inherited ALAS2 defects in treatment with pyridoxine.3 sideroblastic anaemia are similarly likely to Mammals have two ALA synthase genes result from point mutations that modify but (table 2). The X chromosome gene encodes do not abolish enzyme activity. Not all ALAS2 which is expressed only in erythroid families with X linked sideroblastic anaemia cells while that on chromosome 3 encodes a have defects in the ALAS2 gene. Combined housekeeping isoenzyme (ALAS 1) that is use of a highly polymorphic dinucleotide found in all tissues.F6 The C-terminal portion repeat in intron 7 of the gene and other X of ALAS2 (exons 5-11) has 73% sequence chromosome polymorphic markers in linkage identity with ALAS1 and contains the cata- studies has shown that there are at least two lytic domain. Exons 2-4 of the ALAS2 gene X loci for sideroblastic anaemia.26 Overall, encode the N-terminal signal sequence that these studies suggest that analysis of the Departent of directs mitochondrial import and an inter- ALAS2 gene is likely to improve both diagno- Medical vening section that undergoes differential sis and classification of these anaemias and Biochemistry, University of Wales pre-mRNA splicing to generate two isoforms may even reveal a role for somatic mutation College of Medicine, (table 2).6 The functional importance of this of this gene in some acquired forms. Cardiff CF4 4XN pattern of processing has not been estab- G H Elder lished.6 Expression of the ALAS2 gene is Autosomal dominant porphyrias Correspondence to: Professor G H Elder regulated at transcriptional and translational The enzyme defects in most porphyrias are Accepted for publication levels. Its promoter region, in common with inherited in an autosomal dominant manner 17 March 1993 those of the erythroid PBGD,2' ferro- (table 1). The clinical penetrance of these 978 Elder

Table 1 Disorders caused by partial deficiencies ofhaem biosynthetic enzymes exclude exon 2. Translation then proceeds Disorder Enzyme deficiency Inhenitance from a start codon in exon 1 so that the

housekeeping isoenzyme is 17 amino acids J Clin Pathol: first published as 10.1136/jcp.46.11.977 on 1 November 1993. Downloaded from 1 Sideroblastic anaemia ALA synthase (AIAS2) X-linked 2 PBG deficiency porphyria PBG synthase (PBGS) AR longer at the N-terminus than the erythroid 3 Acute intermittent porphyria PBG deaminase (PBGD) AD form. Mutations that affect both isoenzymes 4 Congenital Uroporphyrinogen synthase (UROS) AR 5 Porphyria cutanea tarda Uroporphyrinogen decarboxylase (UROD) see text would thus be predicted to occur in exons 6 Hereditary coproporphyria Coproporphyrinogen oxidase (COPROX) AD 3-15 or in intronic sequences that determine 7 Variegate porphyria Protoporphyrinogen oxidase (PROTOX) AD 8 Erythropoietic protoporphyria (FC) AD the structure or level of expression of the corresponding mRNA. AR: AD: autosomal recessive; autosomal dominant; PBG: porphobilinogen. Three subtypes of AIP can be distin- guished by measurement of erythrocyte PBGD: an uncommon (less than 5% of disorders, however, is low; in each condition families) form in which erythrocyte PBGD is more than 80% of those who inherit the normal with only the ubiquitous isoenzyme genetic defect never have symptoms and most being defective; a form in which the product of these have no detectable biochemical of the mutant allele cross-reacts immuno- abnormality apart from enzyme deficiency. logically with antiserum to normal enzyme The frequency of the genes for these condi- but has absent or substantially impaired cata- tions in the population is sufficiently high for lytic activity (CRIM positive); and CRIM homozygous forms of each disorder to occur negative AIP in which immunoreactivity and without consanguinity127 and for occasional catalytic activity are decreased in parallel. co-inheritance of two separate porphyrias.28 Mutations have been identified in all three subtypes. In the non-erythroid subtype two ACUTE INTERM=ITENT PORPHYRIA mutations that affect the splicing of exon 1 Acute intermittent porphyria (AIP) is the during pre-mRNA processing and thus commonest of the acute hepatic porphyrias impair the formation of mRNA for the ubiq- and at present is the only one that has been uitous isoenzyme only have been found in investigated at the DNA level. It is charac- seven of 10 unrelated patients.29 About 15% terised by life-threatening acute neurovisceral of unrelated patients with AIP have the attacks that are frequently precipitated by CRIM positive subtype. Studies from the drugs, calorie restriction, or alcohol.' Detec- Netherlands and France suggest that about tion of asymptomatic gene carriers so that 75% of these patients have mutations that they can be advised to avoid known lead to replacement of either of two con- precipitants of acute attacks is an important served arginine residues (R167 and R173) in part of the management of AIP families.' 2 exon 10 by glutamine or tryptophan with one AIP is caused by porphobilinogen deami- mutation (G-+A at base position 500; nase (PBGD) deficiency. Enzyme activity is R1 67Q) being more frequent than the close to 50% of normal, reflecting expression others.3031 These mutations severely impair

of the normal gene allelic to the mutant gene. but do not abolish enzyme activity.32 Inter- http://jcp.bmj.com/ As with ALA synthase, there are separate estingly, the only two unrelated cases of erythroid and housekeeping isoenzymes of homozygous AIP that have been reported PBG deaminase (table 2)9; current evidence were both compound heterozygotes for three suggests that there are no tissue specific of the CRIM positive exon 10 mutations,3 isoenzymes for the other enzymes of the suggesting that their frequency may be higher pathway. In contrast to ALA synthase, how- than the prevalence of overt CRIM positive

ever, PBGD is encoded by a single gene AIP would indicate. The structure of PBG on September 27, 2021 by guest. Protected copyright. (table 2) which is transcribed from separate deaminase is highly conserved with over 45% erythroid specific and housekeeping pro- amino acid sequence identity between the moters.910 The erythroid promoter lies in Escherichia coli and human enzymes. X-ray intron 1 and transcription is initiated 5' to analysis of the crystal structure of the E coli exon 2. Translation of erythroid mRNA starts enzyme shows that the arginine residues cor- in exon 3 so that the erythroid isoenzyme responding to these two residues in exon 10 contains sequence encoded by exons 4-15 of human PBGD and a third one in exon 3, and a 3' section of exon 3. RNA for the which is the site of another CRIM positive housekeeping isoenzyme is transcribed from a mutation (R26H) in AIP,34 form salt bridges promoter 5' to exon 1 and then spliced to with the acidic side chains of the dipyrro-

Table 2 Molecular genetics ofhuman haem biosynthesis cDNA in base pairs Enzyme Gene (length ofORF) Expression Chromosome References ALAS1 1920(168) U 3p21 45 ALAS2 22kb, 11 exons 1746(147) E Xpll-21 46 1635(147) E PBGS 990 U 9q34 7 PBGD 1Okb, 15 exons 1032 E llq 81011 1083 U 24-1-q24-2 910 UROS 45kb 795 U 10q25-2-q26-3 12-14 UROD 3kb, 10 exons 1101 U 1p34 1516 COPROX 9 17 PROTOX 14 18 FC 45kb, 11 exons 1269(162) U 18q21-3 1920 ORF: open reading frame. Number of bases encoding N-terminal signal peptides for mitochondrial import are shown in parentheses. U: ubiquitous; E: erythroid cells only. Molecular genetics ofdisorders ofhaem biosynthesis 979

methane cofactor that lies within the cleft Table 3 Human PBG deaminase gene: intragenic between domains 1 and 2.5 CRRIM positive two-allele polymorphisms AIP thus seems to result from mutation of Polymorphism Site

Frequency J Clin Pathol: first published as 10.1136/jcp.46.11.977 on 1 November 1993. Downloaded from residues that are both important for catalysis C/T exon 1 0-63(C) and located deep within the active site cleft so MspI intron 1 0 40 PstI intron 1 0-60 that their substitution does not alter surface ApaLI, SnoI intron 1 0-40 epitopes. BstNI, ScrFI, intron 1 0-55 G/A intron 3 -0-60(G) The commonest subtype, CRIM negative G/T exon 10 0-65(G) AIP, seems to be more heterogeneous than For RFLPs, the frequency is that of the uncut allele. the others. At least 20 base substitutions, MspI, PstI, and ApaLI RFLPs are in complete linkage dis- insertions, or deletions that produce mis- equilibrium. sense, nonsense, or splice defective mutations have been identified.3'3-2 Most have been found in only one or two families but a non- position may change once the full extent of sense mutation in exon 10 (Q198 stop) is the molecular heterogeneity of AIP has been common in Sweden, where it seems to have established and as improved methods for spread through a founder effect from a family rapid screening for large numbers of known originating in Lapland and to explain the high mutations become available. frequency of overt AIP in this country (1 in 1500 in the north).40 About one third of OTHER AUTOSOMAL DOMINANT PORPHYRIAS Dutch families with AIP share an Ri 16Q Porphyria cutanea tarda is a cutaneous mutation, again suggesting a founder effect.42 porphyria that results from decreased activity DNA analysis has an important practical of uroporphyrinogen decarboxylase (UROD) application in AIP for the identification of in the liver. Its pathogenesis is complex but asymptomatic gene carriers. Several stud- one form (type II), which accounts for about ies43-46 have shown its superiority for this 20% of cases, is associated with autosomal purpose over conventional biochemical dominant inheritance of UROD deficiency in techniques, which have a number of draw- all tissues.' More severe UROD deficiency backs.2 At present, the following would seem (activity less than 25% of normal) charac- a reasonable strategy for detecting carriers terises the much rarer, presumed homozy- by DNA techniques. For families in which gous form of type II porphyriac cutanea the mutation is known, methods based tarda, hepatoerythropoietic porphyria (HEP), on polymerase chain reaction (PCR) for which is biochemically heterogeneous.50 Five their detection will give unequivocal point mutations and a deletion of the UROD assignments.2 3434042 Similarly, patients with gene have now been identified in patients the non-erythroid or CRIM positive subtypes, with inherited deficiencies of this enzyme.5" in which certain mutations are common, or Contrary to the anticipated relationship, from areas where one mutation is frequent, mutations identified in patients with HEP

can be rapidly screened for the presence of have not yet been found in those presenting http://jcp.bmj.com/ these mutations.29 30 For those families in with type 11."' For example, the G281E muta- which the mutation is unknown, which at tion, which decreases enzyme stability52 and present means most, two approaches are was present in one Tunisian and five Spanish possible: gene tracking using linkage to families with HEP, was not detected in 12 intragenic polymorphisms43-46 or identifi- type II porphyria cutanea tarda families from cation of the mutation in the proband Spain (Roberts AG, Elder GH, unpublished

followed by direct screening of relatives.3' observations). The UROD defect in type II on September 27, 2021 by guest. Protected copyright. Seven intragenic two allele polymorphisms porphyria cutanea tarda is CRIM negative have been identified in the PBGD gene and it may be that most of the causative (table 3).3 43454748 Three are in complete link- mutations are too severe for homozygotes age disequilibrium and there is partial linkage to survive. Garey et al53 found that five of disequilibrium between others so that the full 22 North American families with type II number of haplotypes is not observed.4549 porphyria cutanea tarda had a splice site Most patients, however, are heterozygous at mutation producing deletion of exon 6 from one allele or more and study of their families mRNA and encoding an inactive, unstable is therefore potentially informative. The main truncated protein. This mutation was not disadvantage is that it is often impossible to present in 25 European families (Roberts AG, establish linkage because not enough Elder GH, unpublished observations), which unequivocally affected relatives are available together with other studies of the frequency for investigation. An alternative but more of mutant uroporphyrinogen decarboxy- laborious approach is to identify the mutation lases,5' suggests extensive molecular hetero- in genomic DNA or cDNA from the family geneity in this group of disorders. under investigation by using a rapid scanning Progress has also been made in under- method, such as denaturing gradient gel standing the molecular basis of erythropoietic electrophoresis (DGGE), to locate the muta- protoporphyria (EPP). This disorder is char- tion, followed by direct sequencing."" acterised by severe photosensitivity; in a few DGGE of seven exons and their flanking patients accumulation of protoporphyrin in sequences after PCR amplification identified hepatocytes leads to irreversible liver failure.' the mutations in 18 of 43 families, suggesting In most families EPP is inherited as an auto- that most mutations would be detected if somal dominant trait with low penetrance,27 investigation was extended to all the exons. but other modes of inheritance have been At present, gene tracking is likely to be the postulated.54 Animal models of the disease, first choice for most laboratories, but the whether occurring naturally in cattle,55 or 980 Elder

produced by ethylnitrosourea mutagenesis in porphyria alleles.65-7 Most patients are com- mice,56 are inherited as autosomal recessive pound heterozygotes and no clear relation- of carriers is between genotypes and the two different traits. Accurate identification ship J Clin Pathol: first published as 10.1136/jcp.46.11.977 on 1 November 1993. Downloaded from important if families are to be counselled phenotypes has yet been established. Homo- adequately. zygotes for the C73R mutation, however, are EPP results from deficiency of ferro- severely affected while patients with a T228M chelatase (table 1). The activity of this mutation of one allele seem to have less enzyme is less than the half-normal that severe disease.67 The infantile form of con- would be expected for an autosomal domi- genital erythropoietic porphyria is probably nant disorder.' 27 Functional ferrochelatase the only type of porphyria for which prenatal may be a homodimer57; if this is so, inter- diagnosis should be available. DNA analysis action between normal and mutant subunits should allow this to be achieved with greater might decrease activity by greater than precision than is currently possible. 50%.7 58 Five point mutations that cause EPP have recently been identified in the fer- The future rochelatase gene. One patient with the rare Several important problems in the pathogene- homozygous form of EPP was a compound sis and management of inherited disorders of heterozygote for two different missense muta- haem biosynthesis now seem likely to be tions.59 In three other families autosomal solved by the application of recombinant dominant inheritance of mutations causing DNA methods. In the acute hepatic por- aberrant splicing606' or an amino acid substi- phyrias, the way towards much more accurate tution leading to decreased activity58 were detection of gene carriers is already clear from found. It thus seems likely that carrier detec- studies on AIP; similar progress for variegate tion in EPP will be complicated by extensive porphyria and hereditary coproporphyria molecular heterogeneity. Linkage analysis in should follow once the relevant genes have EPP families, however, should be facilitated been cloned. The influence of genetic factors by the recent discovery of a dinucleotide in determining phenotypic variation and dif- repeat polymorphism in intron 2 of the ferences between families in the penetrance of gene.62 At present, there is no evidence that the autosomal dominant porphyrias remains particular genotypes are associated with the to be explored. For example, genes that propensity to develop liver disease. directly or indirectly influence the inducibility of ALAS 1 in the liver may be important Autosomal recessive porphyrias determinants of susceptibility to acute por- Two types of porphyria are inherited in an phyria. Finally, transgenic animal experi- autosomal recessive pattern: PBGS(5-amino- ments may help to solve outstanding laevulinate dehydratase) deficiency porphyria problems, such as the mechanism of the acute and congenital erythropoietic porphyria porphyric attack, and lead to new treatments. (CEP) (table 1).

Patients from the four unrelated families 1 Kappas A, Sassa S, Galbraith RA, Nordmann Y. The por- http://jcp.bmj.com/ with PBGS deficiency porphyria that have phyrias. In: Scriver C, Beaudet A, Sly W. Valle D, eds. The metabolic basis of inherited disease. Ann Clin been described show substantial phenotypic Biochem 1990;27:395-412. variation, ranging from failure to thrive in 3 Bottomley SS. The sideroblastic anemias. In: Lee RG, Bithell TC, Foester J, Athens JW, Lufkens JN, eds. infancy through the onset of acute attacks of Wintrobe's clinical haematology. Philadelphia: Lea & porphyria soon after puberty, to subacute Febiger, 1993:852-71. 4 Bishop DF. Two different genes encode o-aminolevulinate polyneuropathy at the age of 63.63 Patients synthase in humans: nucleotide sequences of cDNAs for with the infantile and teenage onset forms the housekeeping and erythroid genes. Nucleic Acids Res on September 27, 2021 by guest. Protected copyright. 1990;18:7187-8. have been shown to be compound heterozy- 5 Cox TC, Bawden MJ, Martin A, May BK. Human ery- gotes for four different missense mutations in throid 5-aminolevulinate synthase: promoter analysis and identification of an iron-responsive element in the the PBGS gene63 64; expression studies show mRNA EMBO J' 1992;10: 1891-902. that three of these substantially decrease 6 Conboy JG, Cox TC, Bottomley SS, et al. Human ery- throid 5-aminolevulinate synthase. J Biol Chem 1992; enzyme activity, but the other, which was 267:18753-8. present in a teenage patient,64 was less severe, 7 Wetmur JG, Bishop D, Cantelmo C, Desnick RJ. Human b-aminolevulinate dehydratase: nucleotide sequence of a suggesting some correlation between genotype and full-length cDNA clone. Proc Nad Acad Sci USA 1986; phenotype in this very rare condition. 83:7703-7. 8 Raich N, Romeo PH, Dubart A, et al. Molecular cloning Congenital erythropoietic porphyria is the and complete primary sequence of human erythrocyte least common of the main types of porphyria porphobilinogen deaminase. Nucleic Acids Res 1986;14: 5955-68. apart from PBGS deficiency. Typically, it 9 Grandchamp B, De Verneuil H, Beaumont C, et al. presents in early infancy with severe skin Tissue-specific expression of porphobilinogen deami- nase. Two isoenzymes from a single gene. EurI Biochem lesions that progress to photomutilation 1987;162: 105-10. which are accompanied by splenomegaly and 10 Chretien S, Dubart A, Beaupain D, et al. Alternative tran- scription and splicing of the human porphobilinogen haemolytic anaemia of variable severity. deaminase gene result either in tissue-specific or in There is also a less severe form with onset housekeeping expression. Proc Nad Acad Sci USA 1988; 85:6-10. after childhood.'27 There are no reports of 11 Namba H, Narahara K, Tsuji Y, Yokoyama Y, Seino Y. both forms occurring in the same family Assignment of human porphobilinogen deaminase to 1lq24 1-+q24-2 by in situ hybridisation and gene which suggests that they may be produced by dosage studies. Cytogenet Cell Genet 1991;57:105-8. different genotypes. Congenital erythropoietic 12 Tsai S-F, Bishop D, Desnick R. Human uroporphyrino- gen III synthase: Molecular cloning, nucleotide porphyria is caused by decreased activity of sequence and expression of a full-length cDNA. Proc UROS (table 1). Eight mutations have been Nal Acad Sci USA 1988;85:7049-53. 13 Warner CA, Yoo HW, Tsai S-F, et al. Congenital erythro- identified in the UROS gene or cDNA in poietic porphyria: characterization of the genomic patients with congenital erythropoietic por- structure and identification of mutations in the uropor- phyrinogen Ill synthase gene. Am J Hum Genet 1990; phyria; one of these (C73R) accounts for 47(supplement):321A. about 20% of congenital erythropoietic 14 Astrin KH, Warner CA, Han-Wook Yoo, et al. Regional Molecular genetics ofdisorders ofhaem biosynthesis 981

assignment of the human uroporphyrinogen m synthase gen deaminase gene in patients with acute intermittent (UROS) gene to chromosome 10q25 2-q-26 3. Hum porphyria by direct sequencing of in vitro amplified Genet 1991;87:18-22. cDNA. Hum Genet 1992;90:12-6. 15 Romeo P-H, Raich N, Dubart A, et al. Molecular cloning 42 X-F, de Rooij F, Lee JS, et High prevalence of Gu al. a J Clin Pathol: first published as 10.1136/jcp.46.11.977 on 1 November 1993. Downloaded from and nucleotide sequence of a complete human uropor- point mutation in the porphobilinogen deaminase gene phyrinogen decarboxylase cDNA. J7 Biol Chem 1986; in Dutch patients with acute intermittent porphyria. 261:9825-31. Hum Genet 1993;91:128-30. 16 Romana M, Dubart A, Beaupain D, et al. Structure of the 43 Llewellyn DH, Elder GH, Kalsheker NA, et al. DNA gene for human uroporphyrinogen decarboxylase. polymorphism of human porphobilinogen deaminase Nucleic Acids Res 1987;15:7343-55. gene in acute intermittent porphyria. Lancet 1987;ii: 17 Grandchamp B, Weil D, Nordmann YN, et al. 706-8. Assignment of the human coproporphyrinogen oxidase 44 Grandchamp BN, Picat C, Mignotte V, et al. Tissue-spe- to chromosome 9. Hum Genet 1983;64:180-3. cific splicing mutation in acute intermittent porphyria. 18 Bissbort S, Hitzeroth HW, Wentzel DP du, et al. Linkage Proc Nate Acad Sci USA 1989;86:661-4. between the variegate porphyria (VP) and the alpha-l- 45 Lee J-S, Lundin G, Lannfelt L, et al. Genetic heterogene- antitrypsin (PI) genes on human chromosome 14. Hum ity of the porphobilinogen deaminase gene in Swedish Genet 1988;79:289-90. families with acute intermittent porphyria. Hum Genet 19 Nakahashi Y, Taketani S, Okuda M, et al. Molecular 1991;87:484-8. cloning and sequence of cDNA encoding human ferro- 46 Kauppinen R, Peltonen L, Palotie A, Mustajoki P. RFLP chelatase. Biochem Biophys Res Comm 1990;173:748-55. analysis of three different types of acute intermittent 20 Taketani S, Inazawa J, Nakahashi Y, et al. Structure of the porphyria. Hum Genet 1990;85:160-4. human ferrochelatase gene. Eur J Biochem 1992;105: 47 Gu XF, Lee J-S, Delfau MH, Grandchamp B. PCR 217-22. detection of a polymorphism at exon 10 of the porpho- 21 Orkin H. Globin gene regulation and switching: circa bilinogen deaminase gene (PBG-D). Nucleic Acids Res 1990. CeU 1990;63:665-72. 1991;19: 1966. 22 Danekar T, Stripecke R, Gray NK, et al. Identification of 48 Picat C, Bourgeois F, Grandchamp B. PCR detection of a a novel iron-responsive element in murine and human C/T polymorphism in exon 1 of the porphobilinogen erythroid -aminolevulinic acid synthase mRNA. deaminase gene (PBGD). Nucleic Acids Res 1991;19: EMBOJ3 1991;10:1903-91. 5099. 23 Haile D, Rouault TA, Harford JB, et al. Cellular regula- 49 Scobie GA, Urquhart AJ, Elder GH, et al. Linkage tion of the iron-responsive element binding protein: disequilibrium between DNA polymorphisms within the Disassembly of the cubane iron-sulfur cluster results in porphobilinogen deaminase gene. Hum Genet 1990; high-affinity RNA binding. Proc Natl Acad Sci USA 85:157-9. 1992;89:11735-9. 50 K6sz6 F, Elder GH, Roberts A, Simon N. Uroporphy- 24 Lathrop JT, Timko MP. Regulation by of mito- rinogen decarboxylase deficiency in hepatoerythro- chondrial protein transport through a conserved amino poietic porphyria: further evidence for genetic acid motif. Science 1993;259:522-5. heterogeneity. BrjDermatol 1990;122:365-70. 25 Cotter PD, Baumann M, Bishop DF. Enzymatic defect in 51 Vemeuil H de, Bourgeois F, Rooij F de, et al. "X-linked" sideroblastic anaemia: molecular evidence Characterization of a new mutation (R292G) and a for erythroid -aminolevulinate synthase deficiency. Proc deletion at the human uroporphyrinogen decarboxylase NaedAcad Sci USA 1992;89:4028-32. locus in two patients with hepatoerythropoietic por- 26 Cox TC, Kozman HM, Raskind W, et al. Identification of phyria. Hum Genet 1992;89:548-52. a highly polymorphic marker within intron 7 of the 52 Verneuil H de, Grandchamp B, Beaumont C, et al. ALAS2 gene and suggestion of at least two loci for X- Uroporphyrinogen decarboxylase structural mutant linked sideroblastic anemia. Hum Mol Genet 1992;1: (Gly 281-+ Glu) in a case or porphyria. Science 1986; 639-41. 234:732-4. 27 Nordmann Y, Deybach JC. Human hereditary porphyrias. 53 Garey JR, Harrison LM, Franklin KF, et al. In: Dailey HA, eds. Biosynthesis of heme and chorophylls. Uroporphyrinogen decarboxylase: a splice site mutation New York: McGraw-Hill, 1990:491-542. causes the deletion of exon 6 in multiple families with 28 Doss M. New dual form of porphyria. Lancet 1988;i: porphyria cutanea tarda. J7 Clin Invest 1990;86: 1416-22. 945-6. 54 Norris PG, Nunn AV, Hawk JLM. Genetic heterogeneity 29 Bourgeois F, Gu X-F, Deybach JC, et al. Denaturing gra- in erythropoietic protoporphyria; a study of the enzy- dient gel electrophoresis for rapid detection of latent matic defect in nine affected families. Invest Dermatol carriers of a subtype of acute intermittent porphyria 1990;95:260-3. with normal erythrocyte porphobilinogen deaminase 55 Straka JG, Hill HD, Krickava JM, et al. Immunochemical activity. Clin Chem 1992;38:93-5. studies of ferrochelatase protein: characterization of the 30 Gu X-F, Rooij F de, Voortman G, et al. High frequency of normal and mutant protein in bovine and human proto-

mutations in exon 10 of the porphobilinogen deaminase porphyria. Am Jf Hum Genet 199 1;48:72-8. http://jcp.bmj.com/ gene in patients with a CRIM-positive subtype of acute 56 Tutois S, Montagutelli X, Da Silva V, et al. Erythropoietic intermittent porphyria. Am J? Hum Genet 1992;51: protoporphyria in the house mouse. A recessive inher- 660-5. ited ferrochelatase deficiency with anemia, photosensi- 31 Gu X-F, de Rooji F, Vooruman G, et al. Detection of tivity and liver disease. Jf Clin Invest 1991;88:1730-6. mutations responsible for acute intermittent porphyria 57 Straka JG, Bloomer JR, Kempner ES. The functional size using denaturing gradient gel electrophoresis. Hum of ferrochelatase determined in situ by radiation inacti- Genet (in press). vation. Jf Biol Chem 1991 ;266:24637-41. 32 Delfau MH, Picat C, de Rooij FWM, et al. Two different 58 Brenner DA, Didier JM, Frazier F, et al. A molecular point G to A mutations in exon 10 of the porphobilino- defect in human protoporphyria. Am Jf Hum Genet responsible for acute intermit- 1992;50:1203-10. gen deaminase gene are on September 27, 2021 by guest. Protected copyright. tent porphyria. Jf Clin Invest 1990;86:1511-6. 59 Lamoril J, Boulechfar S, Verneuil de H, et al. Human 33 Llewellyn DH, Smyth SJ, Elder GH, et al. Homozygous erythropoietic protoporphyria: two point mutations in acute intermittent plorphyria caused by adjacent base the ferrochelatase gene. Biochem Biophys Res Comm transitions in the same codon of the porphobilinogen 1991;18:281-5. deaminase gene. Hum Genet 1992;89:97-8. 60 Nakahashi Y, Fujita H, Taketani S, et al. The molecular 34 Llewellyn DH, Whatley S, Elder GH. Acute intermittent defect of ferrochelatase in a patient with erythropoietic porphyria caused by an arginine to histidine substitution protoporphyria. Proc NadAcad Sci USA 1992;89:281-5. (R26H) in the co-factor binding cleft of porphobilino- 61 Sarkany RPE, Whitcombe DM, Cox TM. Erythropoietic gen deaminase. Hum Mol Genet 1993;2:1315-6. protoporphyria: cloning, sequencing and chromosomal 35 Louie GV, Brownlie PD, Lambert R, et al. Structure of localization of the ferrochelatase gene. Abnormal RNA porphobilinogen deaminase reveals a flexible multi- splicing in an affected family. Br Jf Dermatol 1992; domain polymerase with a single catalytic site. Nature 127(Suppl 40):19. 1992;359:33-40. 62 Whitcombe DM, Cox TM. Dinucleotide repeat polymor- 36 Grandchamp B, Picat C, de Rooij FWM, et al. A point phism at the locus for human ferrochelatas (FECH). mutation G/A in exon 12 of the PBG deaminase gene EMBO Jf (in press). results in exon skipping and is responsible for acute 63 Plewinska M, Thunell S, Holmberg L, et al. 8- intermittent porphyria. Nucleic Acids Res 1989;27: Aminolevulinate dehydratase deficient porphyria: 6637-49. identification of the molecular lesions in a 37 Llewellyn DH, Urquhart A, Scobie G, et al. Molecular severely affected homozygote. Am J7 Genet 1991;49: analysis of acute intermittent porphyria. Biochem Soc 167-74. Trans 1988;16:799-80. 64 Ishida N, Fujita H, Fukuda Y, et al. Cloning and expres- 38 Scobie GA, Llewellyn DH, Urquhart AJ, et al. Acute sion of the defective genes from a patient with b- intermittent porphyria caused by a C-+T mutation that aminolevulinate dehydratase porphyria. 7 Clin Invest produces a stop codon in the porphobilinogen deami- 1992;89: 1431-7. nase gene. Hum Genet 1990;85:631-4. 65 Deybach JC, Verneuil H de, Boulechfar S, et al. Point 39 Delfau MH, Picat C, De Rooij F, et al. Molecular hetero- mutations in the uroporphyrinogen III synthase gene in geneity of acute intermittent porphyria: identification congenital erythropoietic porphyria. Blood 1990:75: of four additional mutations resulting in the CRIM- 1763-5. negative subtype of the disease. Am J7 Hum Genet 1991; 66 Warner CA, Yoo H-W, Roberts AG, Desnick RJ. 49:421-8. Congenital erythropoietic porphyria: identification and 40 Lee J-S, Anvret M. Identification of the most common expression of exonic mutations in the uroporphyrinogen mutation within the porphobilinogen deaminase gene in m synthase gene. 7 Clin Invest 1992;89:693-700. Swedish patients with acute intermittent porphyria. Proc 67 Boulechfar S, DaSilva V, Deybach J-C, et al. NatlAcad Sci USA 1991;88:10912-5. Heterogeneity of mutations in the uroporphyrinogen III 41 Mgone CS, Lanyon WG, Moore MR, Connor JM. synthase gene in congenital erythropoietic porphyria. Detection of seven point mutations in the porphobilino- Hum Genet 1992;88:320-4.