LETTERS TO THE EDITOR

A Spectrum of Novel Mutations in the ProtoporphyrinogenOxidase Gene in 13 Families with Variegate Porphyria

To the Editor:

Variegate porphyria (VP) (OMIM 176000), one of the acutehepatic porphyrias, is caused by a partial de®ciency of proto-porphyrinogen oxidase (PPO) (EC 1.3.3.4), the penultimateenzyme in the porphyrin-heme biosynthetic pathway. Clinically,cutaneous and/or neuropsychiatric symptoms can occur in affectedindividuals (Bickers et al, 1993; Frank and Christiano, 1997). Thecutaneous ®ndings include photosensitivity, increased cutaneousfragility, and blistering on the sun-exposed areas of the skin.Neurologic symptoms can present as acute porphyric attacks,including abdominal pain, muscle weakness, and respiratoryparalysis that can lead to coma and death (Crimlisk, 1997; Frankand Christiano, 1997).

VP is usually inherited in an autosomal dominant fashion withincomplete penetrance. The human PPO cDNA and gene wererecently cloned and mapped to chromosome 1q22±23 (Nishimuraet al, 1995; Taketani et al, 1995), and mutations were identi®ed inseveral families with VP (Deybach et al, 1996; Lam et al, 1996a, b;Meissner et al, 1996; Roberts et al, 1996, 1998; Warnich et al, 1996;Dailey and Dailey, 1997; deRooij et al, 1997; Frank and Christiano,1997; Kauppinen et al, 1997; Frank et al, 1997, 1998a±e, 1999a,2001; Frank and Christiano, 1998; Corrigall et al, 1998; Whatleyet al, 1999).

Here, we studied VP patients of different ethnic origin, ages, andboth genders from 13 unrelated families and 100 unrelated,unaffected control individuals. All patients and family members,their familiar relationship, ethnic origin, and mutations aresummarized in Table I.

EDTA containing blood samples were collected from allindividuals with informed consent. DNA isolation, PCR ampli®-cation, mutation detection/con®rmation, and veri®cation ofpaternity was carried out as previously described in detail (Franket al, 1999a, b).

We identi®ed 11 different mutations in the PPO gene, includingthree nonsense mutations (Q189X, E191X, and Q435X), fourframeshift mutations (1083delT, 1091insA, 915delTG, and1144delGT), three splice site mutations (337 + 1G®A,1290 + 1G®C, and 1292±2 A®G), and one missense mutation(G11S) (Table I). Figure 1 depicts the results of our mutationdetection strategy in Family 3. In heterozygous patients, the wild-type allele was distinguished from the mutant allele using acombination of conformation sensitive gel electrophoresis (CSGE)and automated sequencing on an ABI Prism 310 Genetic Analyzerfrom Applied Biosystems (Perkin Elmer, Foster City, CA). Allmutations reported in this study were initially identi®ed by CSGEanalysis as mutation screening technique. Subsequent automatedsequencing of the PCR fragments showing altered mobilityrevealed the underlying mutations. These mutations were absentin 200 normal chromosomes studied for control purposes.

The molecular heterogeneity of VP is demonstrated by severaldifferent mutations reported in the PPO gene to date, overviewswere previously given by Frank and Christiano (1998) and Whatleyet al (1999).

Interestingly, in the majority of the reported cases of VP,mutations are unique in each individual family; however, recenthaplotyping analyses revealed three exceptions to this rule. Due to afounder effect, R59W is present in approximately 96% of all South

Manuscript received April 28, 2000; revised August 21, 2000; acceptedfor publication January 16, 2001.

Reprint requests to: Dr. Angela M. Christiano, Department ofDermatology, Columbia University, College of Physicians and Surgeons,630 West 168th Street, VC-1526, New York, New York 10032. Email:amc65@columbia.edu

0022-202X/01/$15.00 ´ Copyright # 2001 by The Society for Investigative Dermatology, Inc.

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Table I. VP patients and their family membersa

Family Ethnic origin Mutation

VP 1±01 P American Q435X/±02 S Q435X/±03 S Q435X/±

VP 2 P American 1083delT/±VP 3±01 P American, Indian, 1144delGT/±

02 S German 1144delGT/±03 D 1144delGT/±04 B/P 1144delGT/±05 B ±/±

VP 4 P American 1091insA/±VP 5±01 P American 333 + 1G®A/±

02 S ±/±03 GS ±/±

VP 6 P English, Italian Q189X/±VP 7±01 P Turkish 1291±2 A®G/±

02 D ±/±03 F ±/±

VP 8±01 P American 1290 + 1G®C/±02 S ±/±03 S ±/±04 S ±/±

VP 9±01 P American, German E191X/±02 M E191X/±03 D E191X/±04 D ±/±05 Si ±/±06 B E191X/±07 B E191X/±

VP 10±01 P American, Italian 915delTG/±02 M ±/±03 Si ±/±

VP 11±01 P American, Dutch Q189X/±02 M ±/±03 D ±/±

VP 12±01 P American 1144delGT/±02 M ±/±03 F ±/±04 Si ±/±05 B ±/±

VP 13±01 P American G11S/±02 M ±/±03 F G11S/±

aIndicated are the ethnic origin of each family and the presence/absence of amutation in the PPO gene. P, patient; M, mother; F, father; D, daughter; S, son;Si, sister; B, brother; GS, grandson.

African patients with VP (Meissner et al, 1996; Groenewald et al,1998). Recently, we showed that 1239delTACAC represents anovel founder mutation in the Chilean population, whereasR168H is the ®rst demonstrable hotspot mutation in VP (Franket al, 2000).

Our results re¯ect further exceptions to this rule: Twomutations, Q189X and 1144delGT, respectively, were detectedin four apparently unrelated families (Table I). Interestingly, fouradditional VP families carrying Q189X have been described byother investigators. The same is true for the nonsense mutationQ435X, recently also identi®ed in ®ve VP families from the U.K.(Whatley et al, 1999). Future haplotyping analyses might elucidatethe question if these nonsense mutations are due to a founder effector represent hotspot mutations in the PPO gene.

Systematic molecular analyses and genetic counseling in familieswith VP are very important, in particular because an acuteporphyric attack is a life-threatening condition, with a mortalityranging from 2% to 10% (Lip et al, 1993). As clinical symptoms arenot overt in children before puberty (Kauppinen and Mustajoki,1992), genetic analysis provides the only accurate method toidentify presymptomatic mutation carriers before they are exposedto porphyrinogenic drugs or other factors that might precipitate anacute porphyric attack (Crimlisk, 1997; Moore and Hift, 1997).Particularly in families VP1, 3, and 9, in which several children

were identi®ed as mutation carriers, genetic diagnosis in thepresymptomatic state provides the possibility for early comprehen-sive genetic counseling and should highten the attention of allphysicians treating those patients. Thus, the systematic identi®ca-tion of mutation carriers within VP families is the ®rst and mostimportant step in the prevention of acute porphyric attacks.

The data presented here provide no evidence for the existence ofgenotype-phenotype correlations. We did not observe a moresevere phenotypic expression of certain types of mutations, e.g.those resulting in premature termination codon and therebyabolishing PPO function. In contrast, we also found no correlationbetween the phenotype and the underlying genetic lesion infamilies where several family members carried the same mutation.In family 9 (Fig 1), for example, the severity of clinical symptomswithin mutation carriers ranged from the complete absence of anysymptoms to the most severe combination of extreme photo-sensitivity with recurrent neurologic attacks. These notions aresupported by recently published studies on 104 VP families fromWestern Europe (Whatley et al, 1999).

DNA analysis is clearly the method of choice to con®rm aputative diagnosis of VP, and consequently, to prevent acute attacksin presymptomatic carriers of mutations. Further investigations areunderway to understand unusual mechanisms of inheritance in thisdisease, and to develop gene replacement strategies for this disorder.

Figure 1. Mutation analysis in patient VP 3 and his family members. (A) Pedigree of the family with regard to the occurrence of variegateporphyria (VP). All family members carrying the mutation 1144delGT are indicated by half-®lled squares and circles. (B) Hetero-duplex analysis of family VP 3 and a control individual (C). Note the complex heteroduplex in all individuals carrying 1144delGT. (C) Automatedsequence analysis of the PCR fragment containing exon 11 of the PPO gene. Note the frameshift mutation (1144delGT) of the patient (lower panel)compared with the wild-type sequence of an unrelated control (upper panel).

822 LETTERS TO THE EDITOR THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

We are especially grateful to the patients and their family members for their interest

and cooperation in this study. This study was supported in parts by Grants FR

1315/1±1 (J.F.) from the Deutsche Forschungsgemeinschaft (DFG), START

from the Medizinische FakultaÈt des UniversitaÈtsklinikums der RWTH Aachen, 01

K10 9820F (J.F.) from the Bundesministerium fuÈr Bildung und Forschung

(BMBF), AR 18549 from the NIH (M.B.p.F.), the American Porphyria

Foundation (A.M.C.), and the NIH NIAMS Skin Disease Research Center in

the Department of Dermatology, Columbia University (NIH P30-AR44535)

(D.R.B.).

Jorge Frank,*² Frank K. Jugert,*² Hans F. Merk,* KatrinKalka,³ GuÈnter Goerz,³ Karl Anderson, David R. Bickers,§

Maureen B. Poh-Fitzpatrick,§ Angela M. Christiano§¶*Department of Dermatology and ²Interdisciplinary Center for

Clinical Research (IZKF), University Clinic of the RWTH,Aachen, Germany

³Department of Dermatology, Heinrich Heine University,DuÈsseldorf, Germany

Departments of §Dermatology and ¶Genetics and Development,Columbia University, New York, New York, U.S.A.

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Corrigall AV, Hift RJ, Hancock V, Meissner D, Davids L, Kirsch RE, Meissner PN:Identi®cation and characterisation of a deletion (537delAT) in theprotoporphyrinogen oxidase gene in a South African variegate porphyriafamily. Hum Mutat 12:403±407, 1998

Crimlisk HL: The little imitator-porphyria: a neuropsychiatric disorder. J NeurolNeurosurg Psychiatry 62:319±328, 1997

Dailey HA, Dailey TA: Characteristics of human protoporphyrinogen oxidase incontrols and variegate porphyrias. Cell Mol Biol 43:67±73, 1997

Deybach JC, Puy H, Robreau AM, Lamoril J, Da Silva V, Grandchamp B,Nordmann Y: Mutations in the protoporphyrinogen oxidase gene in patientswith variegate porphyria. Hum Mol Genet 5:407±410, 1996

Frank J, Christiano AM: Genetic research strategies: a review of the acute porphyrias.Retinoids 13:88±92, 1997

Frank J, Christiano AM: Variegate porphyria: past, present and future. Skin PharmacolAppl Skin Physiol 11:310±320, 1998

Frank J, Jugert FK, Kalka K, Goerz G, Merk HF, Christiano AM: Prematuretermination codons in the protoporphyrinogen oxidase gene underlie variegateporphyria. Acta Haematol 98 (Suppl. 1):97, 1997

Frank J, Lam H, Zaider E, Poh-Fitzpatrick M, Christiano AM: Molecular basis ofvariegate porphyria: a missense mutation in the protoporphyrinogen oxidasegene. J Med Genet 35:244±247, 1998a

Frank J, Jugert FK, Kalka K, Goerz G, Merk HF, Christiano AM: Variegateporphyria: identi®cation of a nonsense mutation in the protoporphyrinogenoxidase gene. J Invest Dermatol 110:449±451, 1998b

Frank J, McGrath J, Lam H, Graham RM, Hawk JL, Christiano AM: Homozygousvariegate porphyria: identi®cation of mutations on both alleles of theprotoporphyrinogen oxidase gene in a severely affected proband. J InvestDermatol 110:452±455, 1998c

Frank J, Jugert FK, Breitkopf C, Goerz G, Merk HF, Christiano AM: Recurrentmissense mutation in the protoporphyrinogen oxidase gene underlies variegateporphyria. Am J Med Genet 79:22±26, 1998d

Frank J, Poh-Fitzpatrick MB, King LE Jr, Christiano AM: The genetic basis of`Scarsdale Gourmet Diet' variegate porphyria: a missense mutation in theprotoporphyrinogen oxidase gene. Arch Dermatol Res 290:441±445, 1998e

Frank J, McGrath JA, Poh-Fitzpatrick MB, Hawk JL, Christiano AM: Mutations inthe translation initiation codon of the protoporphyrinogen oxidase geneunderlie variegate porphyria. Clin Exp Dermatol 24:296±301, 1999a

Frank J, Nelson J, Wang X, et al: Erythropoietic protoporphyria: identi®cation ofnovel mutations in the ferrochelatase gene and comparison of biochemicalmarkers versus molecular analysis as diagnostic strategies. J Invest Med 47:278±284, 1999b

Frank J, Aita VM, Ahmad W, Lam H, Wolff C, Christiano AM: Identi®cation of afounder mutation in the protoporphyrinogen oxidase gene in variegateporphyria patients from Chile. Hum Hered, 2000, in press

Groenewald JZ, Liebenberg J, Groenewald IM, Warnich L: Linkage disequilibriumanalysis in a recently founded population: evaluation of the variegate porphyriafounder in South African Afrikaners. Am J Hum Genet 62:1254±1258, 1998

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Kauppinen R, Timonen K, Laitinen E, Kuusisto K, Ahola H, Tenhunen R,Mustajoki P: Molecular genetics and clinical characteristics of variegateporphyria. Acta Haematol 98 (Suppl. 1):96, 1997

Lam H, Dragan L, Tsou HC, et al: Molecular basis of variegate porphyria: Frameshiftmutations in the protoporphyrinogen oxidase gene. J Invest Dermatol 107:144,1996a

Lam H, Dragan L, Tsou HC, et al: Molecular basis of variegate porphyria: a de novoinsertion mutation in the protoporphyrinogen oxidase gene. Hum Genet99:126±129, 1996b

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Meissner PN, Dailey TA, Hift RJ, et al: A R59W mutation in humanprotoporphyrinogen oxidase results in decreased enzyme activity and isprevalent in South Africans with variegate porphyria. Nature Genet 13:95±97,1996

Moore MR, Hift RJ: Drugs in the acute porphyrias: Toxicogenetic diseases. Cell MolBiol 43:89±94, 1997

Nishimura K, Taketani S, Inokuchi H: Cloning of a human cDNA forprotoporphyrinogen oxidase by complementation in vivo of a hem G mutantof Escherichia coli. J Biol Chem 270:8076±8080, 1995

Roberts AG, Puy H, Dailey TA, et al: Molecular characterization of homozygousvariegate porphyria. Hum Mol Genet 7:1921±1925, 1998

Roberts AG, Whatley SD, Dailey TA, Dailey HA, Elder GH: Identi®cation of amutation in the protoporphyrinogen oxidase gene in homozygous variegateporphyria. Hepatology 23:I±94, 1996

deRooij FWM, Minderman G, de Baar E, Wilson JHP, Sinke RJ, Ploos van AmstelJK, te Valde K: Six new protoporphyrinogen oxidase mutations in Dutchvariegate porphyria patients and the R59W mutation in historical perspective.Acta Haematol 98 (Suppl. 1):103, 1997

Taketani S, Inazawa J, Abe T, et al: The human protoporphyrinogen oxidase gene(PPOX): Organization and location to chromosome 1. Genomics 29:698±703,1995

Warnich L, Kotze MJ, Groenewald IM, et al: Identi®cation of three mutations andassociated haplotypes in the protoporphyrinogen oxidase gene in South Africanfamilies with variegate porphyria. Hum Molec Genet 5:981±984, 1996

Whatley SD, Puy H, Morgan RR, et al: Variegate porphyria in Western Europe:Identi®cation of PPOX gene mutations in 104 families, extent of allelicheterogeneity, and absence of correlation between phenotype and type ofmutation. Am J Hum Genet 65:984±994, 1999

Variations in the HCR (Pg8) Gene are Unlikely to be Causalfor Familial Psoriasis

To the Editor:

In a recent report Asumalahti et al identi®ed 12 coding variantswithin gene HCR (Pg8) of the HLA class I region on chromosome

6 (Asumalahti et al, 2000). Two of these variants, C®T transitionsat nucleotide positions of 251 and 269, form an allele (the ``TTallele'') that is strongly associated with psoriasis. Based on this®nding, it was suggested that HCR may have a role in thepathogenesis of psoriasis. We recently reported the ®ne mapping ofthe PSORS1 susceptibility locus to a 60 kb segment of the HLAclass I region, between the HLA-C and corneodesmosin (CDSN)genes (Nair et al, 2000). Using 34 closely spaced microsatellitemarkers, we identi®ed 66 relatively homogeneous clusters ofhaplotypes in 478 families and examined their association with

Manuscript received August 21, 2000; accepted for publicationNovember 16, 2000.

Reprint requests to: Dr. James T. Elder, 3312 Cancer Center andGeriatric Center Building, 1500 E. Medical Center Drive, University ofMichigan, Ann Arbor, MI 48109±0932. Email: jelder@umich.edu

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