Four Novel Mutations in the Gene among Erythropoietic Protoporphyria Patients

Matti H enriksson,* Kaisa Timonen,-j" Pertti Mustajoki,* Hannele Pihlaja,* Raimo Tenhunen ,:I: Leena Peltonen,§ and Raili Kauppinen *

Departments of ' Medicin c, 'I' Dermatology, and :f:C lin ical Chemistry, Uni versity of Hclsin !ci; and §Depa rtrn cnt of I-IU111:111 Molccular Genetics , National Public Health In stitute, Helsinki, Finland

A novel mutation was identified by direct sequencing sequencing of the amplified cDNAs synthesized from of genomic polymerase chain reaction products in total RNA extracted from patients' lymphoblast cell each of four Finnish erythropoietic protoporphyria lines. Because the assays of the ferrochelatase a ctivity families. All four mutations, including two deletions and erythrocyte protoporphyrin identity asymptom­ (751deIGAGAA and the first de IIOVO mutation, atic patients poorly, the DNA-based d e monstration 1122deIT) and two point mutations (286C---') T and of a mutation is the only reliable way to screen 343C---') T), resulted in a dramatically decreased individuals for the dise ase-associated mutation. Key steady-state level of the allelic transcript, since none lVol'd: pOl'pl'Yl'ia. ] Invest Del'llwtol 106:346-350, 1996 of the mutations could he demonstrated by direct

rythropoieti c pro toporphyria (EPP) is an inherited dis­ gene is 1269 bp in size, and only o ne isoform of the enzym e has ease caused by deficient activ ity of ferroch elatase been de m o nstrated (Nakahashi cl ai , 1990). T he gen e is at least 45 (hem e synthase; E.C.4.99.1.1), a mitoch o ndrial en­ kb in size and contains 11 ex ons (Take tani "I ai, 1992). zym e in the biosynthesis pathway (Poh- Fitz­ Since the sequence o f the cD N A and the exon-intron boundaries patrick, 1978). Ferroche latase catalyzes the last step o f of the gene ha ve been available, it h as facilitated the identificati on Ehem e biosynthesis, the ch elation o f ferrous iro n into pro toporphy­ of the mutations amon g EPP patients, and so far 13 mutatio ns in the rin IX to form h em e, and thus overproduction of pro topo rphyrin is ferrochelatase gene , including po in t mutatio ns and de le ti ons, ha ve the m aj o r biochemical abnormality in E PP (Magnus el ai, 196 1) . been identified (Schneider-Yin et ai , 1994; Lamo lil el (/ 1, 1991 ; The disease usuall y m anifests by acute pho to reactions of the skin Sarkany el ai , 1994a,b; Brenner el ai, 1992; Nakahas hi el ai, 1992; already in childhood. Typical sympto m s are stinging pain o r itching 1993a,b; Todd ct ai, 1993; W ang et ai , 1993; M agness cl ai, 1994). w ith subsequent sw elling and erythem a of the sun-exposed ski.n, In this study, we describe fo ur 11 0velmutatio ll s in the fe rrochelatase but chro nic skin ch an ges m ay al so appear (Po h-Fitzpatrick, 1978). gene ide ntifi ed fro m fo ur of the 10 bi ochemicall y and clinicall y w ell In addition, th e skin symptoms m ay be accompanied by potentially characte6 zed EPP fa milies known in Finland. fa tal hepato biliaJ'y complicatio n s in 5-25% o f pati ents (Blo omer et MATEIUAL AND MET HODS al,1 97 5). Inheritance of EPP is considered autosomal dominant with the Patients Twenty-one patients. either symptomatic or asymptomati c. variable penetrance based o n clinical and biochemical analysis, and representi ng 10 known Fin ni sh EPP f:lmilies. and their 11 healthy relatives more recently on molecular gene ti cal studies of EPP patie nts alld wcre in vestigated by DNA analysis. T hc cl inical diagnosis of symptomatic the ir relatives (Lynch and Miedler , 1965; Donaldson el ai, 1967; EPP was based on typical phocosensitivit), of the s!cin, on .increased R eed et ai , 1970; Schneider-Yin et ai, 1994); howeve r, inhetitance erythrocyte protoporpbyrin (Li el nl , 1986) and on low re ticlli ocyte ferro­ chelatase ac tivity (Pasan en "I il l, 1992). T he e"creti on of fccal and urine of an autosomal recessive trait (Went and Klasen , 1984, Lamoril el was dctermincd b), hjgh-perfo rmance liquid chromatograph y (Li ai, 1991; Sarkany el ai, 1994a) or a multiple gene system (No rris el el nl , 1986), and li ver transaminases were measured to evaluate the earl y ai, 1990) h ave also been proposed. T hus, the m o de o f inheritan ce signs of Ij ver dama ge. is still uncertain and m ay in volve differen t m echanism s in individual families. DNA, RNA Extraetion~ , and eDNA Synthesis DN A was e"tracted from whj te blood cells (Higuchi , 1989) and total RNA wa s extractcd from T he ferrochelatase gen e has been assigned to chro m osom e E pste in- BaIT v irus- tran sfectcd lYl11pho biastoid cdl lin es (C hi rw illg et nl. 18(q21.3) based on ill silll h ybridization of the corresponding 1979; Sambrook 1'1 "I, '1989). Complcmentary DNA was synthesizcd frolll cDNA (Brenner et ai, 1992) . I. T he cDNA for the ferrochelatase pati ents ' total lymphobl ast RNA by SuperScri pt II RN asc reverse trall­ scri ptasc (G ibco-BR.L, Ga ithersburg, MD) using random hc"am crs. Manusc rip t received March 13, 1995; revised October 16,1995; accepted Single-strand Conformation Polymorphism (SSCP) Analysis and for publication O ctober 19, :I 995. Sequencing of the Genomic DNA and eDNA Each c"on of the R.eprint requests to: Dr. Raili Kauppinen, Department of Medicin e, ferrochdatase gene was amplified (Mullis and Faloona , 1987) using primers University Hospital of J-/ clsin!ci , Haartmaninkatu 4, 00290 Helsin!ci . Fin­ designed co annea l to th e target sequences of the e"on-intron boundary sites land. of each c"on (Taketani " I "I, 1992) all owing the amplification of eKons 1 !nazawa J, T ake t a l ~ i S, Nakagawa H, In oue K, Mi sa wa S, Abe T : individually. T he DNA (300 ng/sam pl c) was am pli ficd using specifi c Assignment of the human ferrochelatase gene (FCE) to chromosome 18 at prim ers, 30 pmol each in 50 I..d of solutio n. T he te mpera ture profile in the region q21.3. C )'I (),~l'I1 e l Cell cell cl 58:2014. 199 1 (abst. ) polymcrase chain reacti on was 2 min at 94 °C fo r the fir st denatu rin g step.

0022-202X/96/S10.50 • Copyri ght 1996 by T he Society for Iil vestigativ e Dermatology, Inc.

346 VOL. l OG. N O.2 FEUI"tUARY 19% FEIU"tOCH ELATASE MUTATIO NS IN ErP 347

Family 1 Family 2 Mutant Nprmal 5' TAGCTAGC 5' -.- ~~ -- -.- f~ '~ A -..... - A il A -= :=- A del T*!T - :: T -- ..- - C double - - - C m 1 sequence =-- - ."'!- A =-- -- ~ T -:::..- -- - 3' IV -- --

Family 3 Family 4

Mutant Normal 'y TAGCTAGC 5' IJ

III I\ ,T G A Figure 1. Pedigrees of the four EPP families studied.• alld •. affected indi viduals with manifest disease; IiJ and (), asymptomatic individ­ A uals ca rrying the a([ected gene; D and 0 , unallccccd famil y member. D, 1iJ. del GAGAA*/G - and • . male; O . (). and ., female. double sequence

then 30 s at 94°C. 30 s at 52-56°C. and 30 s at 72°C for 30 cycles. For the SSCP anal yses DNA sa mples were radiolabeled during the polymerase chai n reaction by adding 1 }.LC i of a J2p dCTP (Amersham. Buckinghamshire U. K.) into a reaction tube. Single-strand separation gel electrophoresis w ith gels containing 5 or 1 O'X. glycerol was perforn1l!d at room tell1perature. and a gel with no glycerol was run in the cold room (I-la ta el nl, 1990) . For Figure 2. Nucleotide sequences of two patients' genomic DNA sequenci ng the genomic DNA. one of the primer pairs was bi otinylated at demonstrating deletions in the ferrochelatase gene. Upper seq llel/ce. its 5' end, and the polym era se chain reaction products were purified with 11 22dclT in e"on 1 0; lall'eI'seqllel/ce . 751 (or756)delGAGAA in c" on 7. streptavidin-coated lI1i crobcads (Fluoricon; Ide"", Westbrook, M ~E). For scquencin g the cDNA, the amplified samples were run in a low-melting agarose gel, and the fra gl1ll!l1ts were cut out of the gel and purified using Magic PC R. Prep Kit (Promega. Madison. WI). T he products wcre directl y same area in eastern Finland were later found, by a ge n ealogical se quenced usin g the didco"y chain termination method (Sa nger d nl. 1977) . se arc h in church registers, to b e related to each other as well (Fig Amplifica tion and direct sequcncing both with sense and anti-sense primers 1). was repeated at least tl ve independent times from a patient's D NA samples In Family 2, sequen cing of a patient's genomic DNA d e mon- as a rnutatioll site was analyzed . strated a d eletion of nucleotide 1122T in ex o n 10 (Fig 2). This Computer-Based Predictions The hydrophobi city predictions for the lead s to a frameshift and was also predicted to result in a pr e l~l at ur e m utated ferrochc\;'tasc chains werc carried Ollt b), the method of C hou and stop codon 24 amino acid s apart from the d ele tion site (Table I). Fasman (1978) . "nd the secondary structures were predicted by the method SSCP an alysis was performe d in two diffe ren t e lectrophoresis of Hopp and W oods ('198 1) . conditions but no abnormal migration w as o btained. The 11 22delT re presents a de 1/ 0110 event sUl ce it could not b e deplon­ RESULTS strated from the amplifie d genomic DNA of oth er members in the Identified Mutations [n Family 1 (Fig 1), di.rect sequ e ncing of family (Fig 1, Table II). T h e pate rnity of the p atient was con- three patients' amplified genomic DNA demonstrated a d e letio n of five (GAGAAGAGAA) nucle otides in tande m repeat b eguUling from nucleotide 751 (or 7S6)G ill exon 7 (Fig 2). The d e letio n Table I. Characteristics of the Four Mutatiol1s in the cau sed a fram eshift and was pre dicted to result in a premature stop Ferrochelatase Gene cod o n 70 a mino acids apart from the de letion site (Fig 2, Table I). T h e d e letion was confirmed b y restriction cleavage since it also Mutation-Specifi c aboli shed the Mi>o Ji restriction e nzyme site. The SSCP analysis of E"on Mutation Outcomc cDNA R estriction Enzyme exon 7 demonstrated two migrating bands, and the reamplification and direct sequencing of th e b ands c ut o ut individually showed two 3 286C->T Q 96X Avail diffe re nt sequences, th e n o rmal and t he delete d se quence (data not 4 343C->T R115X 7 751dclGAGAA Frame shift Mbo" s bown). T he deletion could be d e monstrated from the patients' 10 I 122dclT ham!! shift relatives (Table II), and the two EPP families originating from the 348 HENRlKSSON ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table II. The DNA Analysis and Biochemical Results of the Members of Four EPP Families

Ferrochclatasc Fecal Activity Erythrocyte Protoporphyrin Mutation (6.0-22.0 pl110l Protoporphyrin (0-130 Ill11 oI/g Screening Photosensitivity hcmc/h/l06 retic) (200 - 1010 nM) dry weight)

Family 1 751delGAGAA Patient 2 (22 y) +" + +" 0.6 23,800' 69 Patient 3 (18 y) + ++ 0.2 26,800 121 Father + 2.2 1,010 NO" Mother 5.1 727 NO Patient 1 (7 y) + +++ 0.6 45,400' 258 Father 11.5 635 ND Mother + 1.4 1,600 NO Grandmother + 2.3 '1. 200 NO Family 2 1l22delT Patient (11 y) + + 1.2 18,100 85 Father 17.3 324 NO Mother 7.4 423 NO Sister 3.0 663 NO Family 3 286C ..... T Patient (9 y) + + + 2.5 28,800 157 Mother 14.1 364 NO Grandfather NO ND NO Family 4 343 C ..... T Patient (33 y) + + ++ 0.4 75 ,600 1204 Father + 2.1 1.400 23 Mother 3.6 423 ND Grandfather' ND + 3.6 4,875 229

tI +. mutation positive; - . mutation negative. r. +, mi1d; ++. moderate; +++. severe. Ii Mean of two (0 five measUrements . •1 ND. not done. , Dead.

finned with 99.94% certainty using DNA fingerprint analysis The results in the assays of reticulocyte ferrochelatase activity (Helminen et ai, 1992). were markedly decreased in every patient having a manifest disease. In Family 3, sequencing of a patient's genomic DNA demon­ Some overlap of the results, however, could be shown among strated a point mutation, 286C~T, in exon 3 (Fig 3; Tables I, II). asymptomatic patients and healthy relatives. The erythrocyte pro­ The mutation was confirmed by cleavage with the A,laH restriction toporphyrin concentration correlated well with the skin symptoms, enzyme. In Family 4, sequencing of a patient's genomic DNA while in the asymptomatic patients the protoporphyrin values were demonstrated a point mutation 343C~T in exon 4 (Fig 3; Tables only marginally elevated. The excretion of fecal protoporphyrin I, II). was increased in a few patients and poorly cOlTelated with the None of the four mutations were found among the other six clinical state. unrelated EPP patients or among 20 unrelated healthy Finnish controls. In addition, only one species of the cDNAs showing DISCUSSION normal sequences could be demonstrated from the patients' ampli­ Four novel mutations including a first de 11.0'/ 0 mutation, two fied cDNA synthesized from total RNA of their lymphoblast lines. deletions and two point mutations, were identified in the ferroche­ Three known polymorphic sites (-23C~T [Nakahashi ct ai, latase gene among families representing 40% of all known EPP 1992], 287A->G [Nakahashi e/ ai, 1990; Sarkany et ai, 1994b], families in Finland. In addition, all four mutations resulted in a 798G~C [Nakahashi ct ai, 1993bJ) were studied among the normal dramatically decreased steady-state level of the al lelic transcript, Finnish population and EPP patients, and the allelic frequencies did since none of the mutations could be demonstrated by direct not differ between the groups studied, except in the case of the sequencing of the ampJjfied cDNAs synthesized from total RNA -23C~T polymorphic site; a T all ele was more frequent among extracted £i'om patients' lymphoblast ceD lines, the technique that EPP patients (total 82 all eles studied, p = 0.01). The haplotyping typically requires some 20-30'Yo of the mutant trallScript to be analysis ofl0 EPP families demonstrated that four potential haplo­ present. Non-sense and frameshift mutations have been demon­ types associated with the symptomatic disease. A 287A~G base strated to associate with a dramatic decrease of cytoplasmic mRNA, change in exon 3 (W96R), an arginine at amino acid 96 was much and it is generall y considered that the closer a termination codon is more common than glutamic acid among the Finnish population, to the 3' end of a gene, the higher the level of the transcript indicating that it is a rare polymorphic site in western populations (Cooper, 1993). Our findings on the termination mutations in the (Lamoril el ai, 1991; Nakahashi ct ai, 1990; Sarkany el ai, 1994b). ferrochelatase gene, however, were not in complete accordance Clinical an4 Biochemical Characteristics of Patients with with this" 5' -3' positional effect." The reduction of the level of Identified Mutations The results of the biochemical assays of mRNA can be affected by transcription rate, processing, or trans­ the patients and their relatives are presented in Table II. Seven port of RNA to cytoplasm; thus, several mechanisms can be patients had manifest EPP. Cutaneous photosensitivity occurred responsible for the deficiency of the mutated mRNA species already in infancy in the case of the severe clinical phenotype, and (Cooper, 1993). None of the mutations were found in the ferro­ in early childhood in the case of moderate or mild disease form. The chelatase pseudogene (Whitcom be cf ai, 1994) excluding erroneous intensity and onset of photosensitivity varied within the families, conclusions of the results. and showed no clear mutation-specific correlation. No signs ofliver EPP usually has an autosomal dominant pattern of inheritance compli cations were found in any of the patients. with variable penetrance, although some authors have suggested VOL. 106, NO. 2 FEI3 I"UAR Y 19% FEKROCH ELATASE MUTATIONS IN EPP 349

Mutant Normal and exon-intron bo wldari es of their genomic DNA was sequenced. Moreover, regarding Fa mily 1., w hich has three patients in two TAGCTAGC separate branches of the pedigree, it would be very unlikely to have 5' additionall11utations in different branches simultaneously, unless an additional common rearrangement would affect the felTochelatase T activity. G All mutations have been fa mily specific, and thus EPP has been G demonstrated to be heterogeneous at the m o lecular level despite of A the relative uniformity of the c1inica] phenotype. AU om' symptom­ C atic patients with different mutations have had characteristic pho­ C tosensitivity with no signs of li ver fai lure. For the present, it seem s to be difti cult to predict the onset or severity of cutaneous o r Li ver G disease based only on a mutation analysis beca use of c1 inica.l variety A within the famili es and the small number of the patients analyzed. G More patient data has to be analyzed fo r the final conclusions of the A genotype-phenotype correlation of EPP. 3' T he dramatic decrease in the ferrochelatase activity and high erythrocyte protoporphyrins are usually characteristic in symptom­ atic EPP patients, but the biochemical m ethods poorly recognize asymptomatic individuals carrying dIe aftccted gene. Thus, the demonstrati on of a mutatio n in the ferrochelatase gene is conve­ nient and the o nly reli abl e way to screen asymptomati c individuals in fa milies whose mutation has been identified.

T h;s slIId y (/Ins SJlpported b)' grnJltsji'Ol ll t.he Emil A nlto ll clI Fo " ",fnt;(lII, III(' Pnlll" Mutant Normal FO II"dl1licHl, ,he IIlSlnllltCII,mi lllJl R('scafch FOIllH/micHI, 'he AradeHl)' oj Fi"lnlld nnd TAGCTAGC the UII; ClCfS ;/Y (~r He!s;lIk;.

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