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Four Novel Mutations in the Ferrochelatase Gene Among Erythropoietic Protoporphyria Patients

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Four Novel Mutations in the Ferrochelatase Gene Among Erythropoietic Protoporphyria Patients Four Novel Mutations in the Ferrochelatase 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 heme 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 porphyrins 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).
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