Haplotype Analysis in Determination of the Heredity of Erythropoietic Protoporphyria among Swiss Families
2001; Elsevier BV; Volume: 117; Issue: 6 Linguagem: Inglês
10.1046/j.0022-202x.2001.01604.x
ISSN1523-1747
AutoresMartin Hergersberg, Jean‐Charles Deybach, Xiaoye Schneider‐Yin, Urszula B. Rüfenacht, Cécile Schnyder, Elisabeth I. Minder,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoDefects in the human ferrochelatase gene lead to the hereditary disorder of erythropoietic protoporphyria. The clinical expression of this autosomal dominant disorder requires an allelic combination of a disabled mutant allele and a low-expressed nonmutant allele. Unlike most other erythropoietic protoporphyria populations, mutations identified among Swiss erythropoietic protoporphyria families to date have been relatively homogenous. In this study, genotype analysis was conducted in seven Swiss erythropoietic protoporphyria families, three carrying mutation Q59X, two carrying mutation insT213, and two carrying mutation delTACAG580-584. Three different haplotypes of five known intragenic single nucleotide polymorphisms, namely -251 A/G, IVS1–23C/T, 798 G/C, 921 A/G, and 1520C/T, were identified. Each haplotype was shared by families carrying an identical mutation in the ferrochelatase gene indicating a single mutation event for each of the three mutations. These mutations have been present in the Swiss erythropoietic protoporphyria population for a relatively long time as no common haplotypes of microsatellite markers flanking the ferrochelatase gene were found, except of two conserved regions, telomeric of the insT213 allele and centromeric of the delTACAG580-584 allele, each with a size > 3 cM. Among the nonmutant ferrochelatase alleles, patients from six erythropoietic protoporphyria families shared a common haplotype [-251G; IVS1–23T] of the first two single nucleotide polymorphisms. An exception was the haplotype [-251 A; IVS1–23C] identified in the index patient of one erythropoietic protoporphyria family. These results supported the recent findings that the low expressed allele is tightly linked to a haplotype [-251G; IVS1–23T] of two intragenic single nucleotide polymorphisms in the ferrochelatase gene. Defects in the human ferrochelatase gene lead to the hereditary disorder of erythropoietic protoporphyria. The clinical expression of this autosomal dominant disorder requires an allelic combination of a disabled mutant allele and a low-expressed nonmutant allele. Unlike most other erythropoietic protoporphyria populations, mutations identified among Swiss erythropoietic protoporphyria families to date have been relatively homogenous. In this study, genotype analysis was conducted in seven Swiss erythropoietic protoporphyria families, three carrying mutation Q59X, two carrying mutation insT213, and two carrying mutation delTACAG580-584. Three different haplotypes of five known intragenic single nucleotide polymorphisms, namely -251 A/G, IVS1–23C/T, 798 G/C, 921 A/G, and 1520C/T, were identified. Each haplotype was shared by families carrying an identical mutation in the ferrochelatase gene indicating a single mutation event for each of the three mutations. These mutations have been present in the Swiss erythropoietic protoporphyria population for a relatively long time as no common haplotypes of microsatellite markers flanking the ferrochelatase gene were found, except of two conserved regions, telomeric of the insT213 allele and centromeric of the delTACAG580-584 allele, each with a size > 3 cM. Among the nonmutant ferrochelatase alleles, patients from six erythropoietic protoporphyria families shared a common haplotype [-251G; IVS1–23T] of the first two single nucleotide polymorphisms. An exception was the haplotype [-251 A; IVS1–23C] identified in the index patient of one erythropoietic protoporphyria family. These results supported the recent findings that the low expressed allele is tightly linked to a haplotype [-251G; IVS1–23T] of two intragenic single nucleotide polymorphisms in the ferrochelatase gene. denaturing gradient gel electrophoresis ferrochelatase single nucleotide polymorphism Erythropoietic protoporphyria (EPP, MIM 177000) is a genetic disorder of porphyrin metabolism caused by a partial deficiency of ferrochelatase activity. Ferrochelatase (FECH; EC 4.99.1.1), the last enzyme of the heme biosynthetic pathway, catalyzes the insertion of Fe2+ ion into protoporphyrin IX to form heme (Kappas et al., 1995Kappas A. Sassa S. Galbraith R.A. Nordmann Y. The porphyrias.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. McGraw-Hill, New York1995: 2139-2141Google Scholar). Defective ferrochelatase leads to the accumulation of protoporphyrin IX, and occurs mainly in the red blood cells. Clinically, an excess amount of free protoporphyrin deposited in the skin causes an extremely painful photosensitivity in EPP patients that starts in early childhood. In addition to cutaneous photosensitivity, a small percentage (<2%) of patients develop hepatobiliary complication as a result of the toxic effect of protoporphyrin on the liver (Bloomer et al., 1998Bloomer J. Bruzzone C. Zhu L. et al.Molecular defects in ferrochelatase in patients with protoporphyria requiring liver transplantation.J Clin Invest. 1998; 102: 107-114Crossref PubMed Scopus (48) Google Scholar). The human FECH gene localized on chromosome 18q contains 11 coding exons and has a size of approximate 45 kb; the entire cDNA as well as partial genomic sequences have been published (Nakahashi et al., 1990Nakahashi Y. Taketani S. Okuda M. Inoue K. Tokunaga R. Molecular cloning and sequence analysis of cDNA encoding human ferrochelatase.Biochem Biophys Res Commun. 1990; 173: 748-755Crossref PubMed Scopus (130) Google Scholar;Whitcombe et al., 1991Whitcombe D. Carter N. Albertson D. Smith S. Rhodes D. Cox T. Assignment of the human ferrochelatase gene (FECH) and a locus for protoporphyria to chromosome 18q22.Genomics. 1991; 11: 1152-1154Crossref PubMed Scopus (61) Google Scholar;Taketani et al., 1992Taketani S. Inazama J. Nakahashi Y. Abe T. Tokunaga R. Structure of the human ferrochelatase gene exon/intron gene organization and location of the gene to chromosome 18.Eur J Biochem. 1992; 205: 217-222Crossref PubMed Scopus (143) Google Scholar). Mutations in the human FECH gene have been shown to be associated with the decrease in ferrochelatase activity in EPP. To date, over 60 different FECH gene mutations have been published in EPP patients from various countries and ethnic backgrounds (Schneider-Yin et al., 2000aSchneider-Yin X. Gouya L. Meier A. Deybach J. Minder E.I. New insights into the pathogenesis of erythropoietic protoporphyria and their impacts on patient care.Eur J Pediatr. 2000; 159: 719-725Crossref PubMed Scopus (56) Google Scholar). The majority of the mutations are family specific with a few exceptions of mutations being shared by a limited number of EPP families. In Ireland, a single base pair deletion (delG40) was identified in three EPP families (Todd et al., 1993Todd D. Hughes A. Ennis K. et al.Identification of a single base pair deletion (40 del G) in exon 1 of the ferrochelatase gene in patient with erythropoietic protoporphyria.Hum Mol Genet. 1993; 2: 1495-1496Crossref PubMed Scopus (30) Google Scholar). In the U.S.A, mutations K379X and IVS1 + 5, g→a (del exon 1), were each shared by four EPP families (Wang et al., 1999Wang X. Yang L. Kurtz L. et al.Haplotype analysis of families with erythropoietic protoporphyria and novel mutation of the ferrochelatase gene.J Invest Dermatol. 1999; 113: 87-92Crossref PubMed Scopus (18) Google Scholar). In Switzerland, recurrence of FECH gene mutations seems to be a rather frequent phenomenon. In the past, we have identified a total of five different mutations in the FECH gene of 13 Swiss EPP families. Four of them, namely mutations Q59X, insT213, delTACAG580-584, and del TG899-900, were found in more than one apparently unrelated family (Rüfenacht et al., 1998aRüfenacht U. Gouya L. Schneider-Yin X. et al.Systematic analysis of molecular defects in the ferrochelatase gene from patients with erythropoietic protoporphyria.Am J Hum Genet. 1998; 62: 1341-1352Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Interestingly, mutation delTACAG580-584 has also been detected in EPP patients from France and the U.S.A. (Bloomer et al., 1998Bloomer J. Bruzzone C. Zhu L. et al.Molecular defects in ferrochelatase in patients with protoporphyria requiring liver transplantation.J Clin Invest. 1998; 102: 107-114Crossref PubMed Scopus (48) Google Scholar;Rüfenacht et al., 1998aRüfenacht U. Gouya L. Schneider-Yin X. et al.Systematic analysis of molecular defects in the ferrochelatase gene from patients with erythropoietic protoporphyria.Am J Hum Genet. 1998; 62: 1341-1352Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In general, molecular defects underlining EPP are heterogeneous, although mutations identified among patients with liver complications share a common feature of producing a truncated enzyme. These so-called “null allele” mutations consist of frameshifts and nonsense mutations. The association between null allele mutations and liver complications in EPP is statistically significant, p <0.05 (Schneider-Yin et al., 2000aSchneider-Yin X. Gouya L. Meier A. Deybach J. Minder E.I. New insights into the pathogenesis of erythropoietic protoporphyria and their impacts on patient care.Eur J Pediatr. 2000; 159: 719-725Crossref PubMed Scopus (56) Google Scholar). The heredity of EPP has a complex pattern and cannot be simply defined as either autosomal dominant or autosomal recessive inheritance. In the majority of EPP families, a single mutation is the only identifiable molecular defect in the FECH gene. Among all carriers of a FECH gene mutation, less than 10% of the individuals will develop the clinical symptoms of EPP, the majority remaining asymptomatic throughout their lives. Various studies have shown that, at the level of enzyme activity, symptomatic patients will exhibit a value 10%-30% of normal, whereas asymptomatic carriers will have approximately 50% of normal enzyme activity (Deybach et al., 1986Deybach J.C. Da Silva V. Pasquier Y. Nordmann Y. Ferrochelatase in human erythropoietic protoporphyria: the first case of a homozygous form of the enzyme deficiency.in: Nordmann Y. Porphyrins and Porphyrias. John Libbey, Paris1986: 173-193Google Scholar;Norris et al., 1990Norris P. Nunn A. Hawk J. Cox T. Genetic heterogeneity in erythropoietic protoporphyria. a study of the enzymatic defect in nine affected families.J Invest Dermatol. 1990; 95: 260-263Abstract Full Text PDF PubMed Google Scholar). The recent work by Gouya and colleagues has led to the disclosure of the low expression mechanism – a step forward towards the understanding of the low clinical penetrance in EPP (Gouya et al., 1996Gouya L. Deybach J.C. Lamoril J. et al.Modulation of the phenotype in dominant erythropoietic protoporphyria by low expression of the normal ferrochelatase gene.Am J Hum Genet. 1996; 58: 292-299PubMed Google Scholar;Gouya et al., 1999Gouya L. Puy H. Lamoril J. et al.Inheritance in erythropoietic protoporphyria: a common wild-type ferrochelatase allelic variant with low expression accounts for clinical manifestation.Blood. 1999; 93: 2105-2110PubMed Google Scholar). Among the patients of EPP families they studied, those with lower than 50% of normal ferrochelatase activity resulted from a reduced mRNA transcription of the nonmutant FECH allele. Two single nucleotide polymorphisms, -251 A/G in the promoter region and IVS1–23C/T, have been shown to be tightly linked to the low expressed FECH allele (Gouya et al., 1999Gouya L. Puy H. Lamoril J. et al.Inheritance in erythropoietic protoporphyria: a common wild-type ferrochelatase allelic variant with low expression accounts for clinical manifestation.Blood. 1999; 93: 2105-2110PubMed Google Scholar). To explore the unique feature of recurrent FECH gene mutations in the Swiss EPP population, haplotype analyses using both intragenic single nucleotide polymorphisms (SNP) and microsatellite markers were conducted among seven EPP families carrying three frequent mutations. The data obtained from this study indicated common ancestral alleles for each of the three mutations, Q59X, insT213, and delTACAG580-584. In addition, the role of two intragenic SNP (-251 A/G and IVS1–23C/T) on the clinical penetrance of EPP was examined in this EPP cohort. A total of 31 individuals from seven unrelated Swiss EPP families, including 11 patients, 11 asymptomatic mutation carriers, and nine healthy individuals, were studied. Peripheral blood samples were collected from these individuals with informed consent. The study design was approved by the ethical committee of Stadtspital Triemli, Zürich. Among the seven EPP families, three families (I, II, and III) carried mutation Q59X, two families (IV and V) carried mutation insT213, and two families (VI and VII) carried mutation delTACAG580-584 in the FECH gene, as was previously published (Schneider-Yin et al., 1994Schneider-Yin X. Schäfer B. Möhr P. Burg G. Minder E.I. Molecular defect in erythropoietic protoporphyria with terminal liver failure.Hum Genet. 1994; 93: 711-713Crossref PubMed Scopus (28) Google Scholar,Schneider-Yin et al., 1995Schneider-Yin X. Schäfer B. Tonz O. Minder E.I. Human ferrochelatase: a novel mutation in patients with erythropoietic protoporphyria and an isoform caused by alternative splicing.Hum Genet. 1995; 95: 391-396Crossref PubMed Scopus (18) Google Scholar;Rüfenacht et al., 1998aRüfenacht U. Gouya L. Schneider-Yin X. et al.Systematic analysis of molecular defects in the ferrochelatase gene from patients with erythropoietic protoporphyria.Am J Hum Genet. 1998; 62: 1341-1352Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, Rüfenacht et al., 1998bRüfenacht U. Schneider-Yin X. Schäfer B. et al.Rapid molecular diagnosis of erythropoietic protoporphyria among Swiss patients.Clin Chem Laboratory Med. 1998; 36: 763-765Crossref PubMed Scopus (5) Google Scholar). Five known intragenic SNP in the human FECH gene, namely -251 A/G in the promoter region, IVS1–23C/T, 798 G/C in exon 7, 921 A/G in exon 9, and 1520C/T in the 3′-UTR, were analyzed in all members of the seven EPP families. In addition, -251 A/G and IVS1–23C/T were determined in 32 control samples. Genomic DNA was isolated from peripheral blood of all subjects using a QIAamp Blood kit (Qiagen, Germany). To analyze -251 A/G, the promoter region of the FECH gene was amplified by using a sense primer 5′-CCG TCC CTC CAA GAA ATG and an antisense primer 5′-GGT GTC CGC CCA GCA GTG. The 574 bp product was digested with restriction enzyme Alu I (Boehringer Mannheim, Germany). Partial sequence of intron 1 was amplified by using a sense primer 5′-TTA CCT GCC TGC AGA GAA ATC A and an antisense primer 5′-GCT GGG CTG TTT CTG TGG TG for determination of IVS1–23C/T. The 235 bp product was digested with restriction enzyme Cac8 I (New England Biolabs, U.K.). Conditions for polymerase chain reaction (PCR) amplifications of exon 7 and 9 of the FECH gene and the subsequent denaturing gradient gel electrophoresis (DGGE) analysis of exon 9 have been previously described (Rüfenacht et al., 1998aRüfenacht U. Gouya L. Schneider-Yin X. et al.Systematic analysis of molecular defects in the ferrochelatase gene from patients with erythropoietic protoporphyria.Am J Hum Genet. 1998; 62: 1341-1352Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Following the PCR, the 220 bp product of exon 7 was digested with restriction enzyme Nla III (New England Biolabs). All enzyme digests were analyzed in a 3% agarose gel. The 3′-UTR of the FECH gene was amplified by PCR using primers as described byGouya et al., 1996Gouya L. Deybach J.C. Lamoril J. et al.Modulation of the phenotype in dominant erythropoietic protoporphyria by low expression of the normal ferrochelatase gene.Am J Hum Genet. 1996; 58: 292-299PubMed Google Scholar. The PCR product was analyzed by DGGE on a D GENE System (Bio-Rad, Hercules, CA) with a linear denaturant gradient of 13%-43%. The DGGE gel (16 × 16 cm) was run at 60°C for 3 h under a constant voltage of 120 V. In addition to the intragenic SNP, a total of nine microsatellite markers were selected for haplotype analysis. Most of the microsatellites were chosen from the Marshfield map (Broman et al., 1998Broman K. Murray J. Sheffield V. White R. Weber J. Comprehensive human genetic maps. individual and sex-specific variation in recombination.Am J Hum Genet. 1998; 63: 861-869Abstract Full Text Full Text PDF PubMed Scopus (893) Google Scholar) for maximum informativity. These markers and additional polymorphisms are located in the genomic region of the FECH gene in the order of cen-D18S69-D18S1152-D18S41-D18S858-FECH-D18S381-D18S77-D18S1144-D18S1155-D18S64-qter in the LDB map (Collins et al., 1996Collins A. Frezal J. Teague J. Morton N.E. A metric map of humans: 23,500 loci in 850 bands.Proc Natl Acad Sci USA. 1996; 93: 14771-14775Crossref PubMed Scopus (243) Google Scholar). The marker heterozygosities and marker distances are given in Table I. PCR primers were either purchased (Research Genetics, Huntsville, AL) or synthesized with the available sequence information. PCR was performed using standard conditions. After a 5 min heat denaturation at 95°C, the PCR products were separated on 4%-6% (depending on the size of the alleles) polyacrylamide sequencing gels of 17 × 50 cm (BioRad, Hercules, CA) under a constant power of 50 W for 1.5–2.5 h (depending on the size of the alleles). DNA fragments were visualized by silver staining (Haider et al., 2000Haider N. Jacobson S. Cideciyan A. et al.Mutation of a nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate.Nat Genet. 2000; 24: 127-131Crossref PubMed Scopus (365) Google Scholar). Arbitrary numbers were assigned to different alleles in the analyzed population, beginning with the largest allele.Table IHaplotypes of the mutant FECH alleles among patients and asymptomatic carriers from EPP familiesFamilyQ59Xins T213delTACAG580-584LocusaMarker order, heterozygosity, and marker distance were obtained from both LDB and GDB (see Materials and Methods). Heterozygosity and map position (cM) are given in parentheses. The FECH gene is located at 63.76 cM.IIIIIIIVVVIVIID18S69(0.78; 60.67 cM)3423322D18S1152(0.80; 62.20 cM)55441,444D18S41(0.77; 62.76 cM)2224222D18S858(0.77; 62.91 cM)5455366-251 A/G(promoter)GGGAAAAIVS1-23 C/T(intron 1)TTTCCCC798 G/C(exon 7)CCCGGGCC921 G/A(exon 9)GAGGGGAA1520 C/T(3′-UTR)CCCCCCCD18S381(0.62; 63.80 cM)6644432D18S977(0.91; 64.93 cM)2,6661166D18S1144(0.85; 65.27 cM)887,121010107D18S1155(0.67; 65.58 cM)11,434444D18S64(0.74; 67.28 cM)53443,455a Marker order, heterozygosity, and marker distance were obtained from both LDB and GDB (see Materials and Methods). Heterozygosity and map position (cM) are given in parentheses. The FECH gene is located at 63.76 cM. Open table in a new tab Allelic characterization of five SNP in the FECH gene is shown in Figure 1. A 574 bp DNA fragment in the promoter region bearing the A/G dimorphism at position -251 was amplified by PCR. The presence of A or G at -251 position was verified by Alu I digestion of the PCR product. Homozygous A/A appeared as three bands of 379 bp, 153 bp, and 42 bp, whereas homozygous G/G appeared as two bands (421 and 153 bp) on the agarose gel. The C/T dimorphism at position -23 near the intron 1-exon 2 junction was characterized by Cac 8I digestion of PCR-amplified genomic DNA. The PCR product was cut into three fragments of 117 bp, 108 bp, and 10 bp in the case of homozygous C/C, and was cut into two fragments of 225 bp and 10 bp in the case of homozygous T/T. A 220 bp fragment spanning over exon 7 of the FECH gene was subjected to Nla III digestion to determine the status of 798 G/C dimorphism. The 220 bp fragment remained intact if a G was present on both alleles. The presence of C at position 798 introduced a Nla III site. The PCR product from a C/C homozygote was therefore digested into two fragments of 127 bp and 93 bp by the enzyme. SNP 921 A/G and 1520 C/T, located in exon 9 and the 3′-UTR of the FECH gene, respectively, were characterized by DGGE analysis. As shown in Figure 1, both 921 G/G and A/A homozygotes appeared as a single band, but with different migration rates in the DGGE gel. The A/G heterozygote showed a characteristic pattern of quadruple bands in the gel. In the case of the 1520 C/T dimorphism, the C/T heterozygote exhibited a pattern of double bands, whereas the single bands from both C/C and T/T homozygotes differed by their migration rates in the DGGE gel. Mutations in the FECH gene of symptomatic patients among seven EPP families were identified by previous sequencing analysis (Schneider-Yin et al., 1994Schneider-Yin X. Schäfer B. Möhr P. Burg G. Minder E.I. Molecular defect in erythropoietic protoporphyria with terminal liver failure.Hum Genet. 1994; 93: 711-713Crossref PubMed Scopus (28) Google Scholar,Schneider-Yin et al., 1995Schneider-Yin X. Schäfer B. Tonz O. Minder E.I. Human ferrochelatase: a novel mutation in patients with erythropoietic protoporphyria and an isoform caused by alternative splicing.Hum Genet. 1995; 95: 391-396Crossref PubMed Scopus (18) Google Scholar;Rüfenacht et al., 1998aRüfenacht U. Gouya L. Schneider-Yin X. et al.Systematic analysis of molecular defects in the ferrochelatase gene from patients with erythropoietic protoporphyria.Am J Hum Genet. 1998; 62: 1341-1352Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Other family members were screened for the family-specific mutation by either DGGE or restriction analyses to identify asymptomatic mutation carriers (Rüfenacht et al., 1998aRüfenacht U. Gouya L. Schneider-Yin X. et al.Systematic analysis of molecular defects in the ferrochelatase gene from patients with erythropoietic protoporphyria.Am J Hum Genet. 1998; 62: 1341-1352Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, Rüfenacht et al., 1998bRüfenacht U. Schneider-Yin X. Schäfer B. et al.Rapid molecular diagnosis of erythropoietic protoporphyria among Swiss patients.Clin Chem Laboratory Med. 1998; 36: 763-765Crossref PubMed Scopus (5) Google Scholar). The statuses of patient, carrier, and unaffected family member are indicated in the pedigrees in Figure 2. Based on this information, haplotypes featured by five intragenic SNP were defined in five pedigrees according to Mendelian inheritance. In families I and III, however, 798 G/C could not be aligned with other markers because all tested individuals from these families were heterozygous G/C. As presented in Figure 2, among all 62 alleles from 31 tested individuals, including patients, carriers, and healthy relatives of the seven EPP families, -251 A was found invariably in cis to IVS1–23C, and -251G was at all times in cis to IVS1–23T. Haplotypes of the mutated FECH alleles from the seven EPP families carrying three different FECH mutations are illustrated separately in Table I. Each of the three mutations was found to cosegregate with one particular haplotype of the five intragenic SNP in all families carrying the respective mutations. Families I, II, and III, carrying the Q59X mutation, shared a common haplotype of [G-T-C-G-C]. Families IV and V, both carrying the insT213 mutation, shared a common haplotype of [A-C-G-G-C]. Families VI and VII, both carrying the delTACAG580-584 mutation, shared a common haplotype of [A-C-C-A-C]. As, as stated above, the nonmutant FECH allele determines the clinical outcome of EPP, haplotypes of patients were compared with those of the asymptomatic carriers. As shown in both Figure 2 and Table II, the nonmutant FECH allele in 10 EPP patients from six families exhibited a common haplotype of [-251G; IVS1–23T] at the first two SNP. Only one patient in family V showed a haplotype of [-251 A; IVS1–23C] in the nonmutant FECH allele. In contrast, the nonmutant FECH allele of all 11 asymptomatic carriers in this study cohort featured the haplotype [-251 A; IVS1–23C] (Figure 2, Table III). As described above, genotype analysis using intragenic SNP indicated identical haplotypes for the three most common FECH gene mutations in Switzerland. To further investigate this phenomenon, nine microsatellite markers were analyzed in all members of the seven EPP families. The microsatellites flank the FECH gene on chromosome 18q22 and span ≈6.6 cM. In general, the results showed that no extended conserved haplotypes were associated with the FECH gene mutations in the analyzed genomic areas except for two regions. As shown in Table I, one conserved telomeric region of the insT213 allele spans ≈3.5 cM; the other conserved centromeric region of the delTACAG580-584 allele spans ≈3.1 cM. Various haplotypes in the extended genomic regions were observed among the seven nonmutant alleles from EPP patients Table II. In particular, no apparent similarities were found among the six microsatellite haplotypes (from families I to VI) that were linked to the common intragenic SNP haplotype of [-251G; IVS1–23T]. Based on the available genetic information, mutations in the human FECH gene are typically heterogeneous or family specific. Among the few exceptions, mutations in the Swiss EPP families have been found to be relatively homogenous (Schneider-Yin et al., 1994Schneider-Yin X. Schäfer B. Möhr P. Burg G. Minder E.I. Molecular defect in erythropoietic protoporphyria with terminal liver failure.Hum Genet. 1994; 93: 711-713Crossref PubMed Scopus (28) Google Scholar,Schneider-Yin et al., 1995Schneider-Yin X. Schäfer B. Tonz O. Minder E.I. Human ferrochelatase: a novel mutation in patients with erythropoietic protoporphyria and an isoform caused by alternative splicing.Hum Genet. 1995; 95: 391-396Crossref PubMed Scopus (18) Google Scholar;Rüfenacht et al., 1998aRüfenacht U. Gouya L. Schneider-Yin X. et al.Systematic analysis of molecular defects in the ferrochelatase gene from patients with erythropoietic protoporphyria.Am J Hum Genet. 1998; 62: 1341-1352Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, Rüfenacht et al., 1998bRüfenacht U. Schneider-Yin X. Schäfer B. et al.Rapid molecular diagnosis of erythropoietic protoporphyria among Swiss patients.Clin Chem Laboratory Med. 1998; 36: 763-765Crossref PubMed Scopus (5) Google Scholar). In this study, we carried out genotype analysis in order to test the hypothesis that families carrying an identical FECH mutation are derived from a common ancestor. This hypothesis was verified insofar as an intragenic SNP haplotype was conserved among analyzed patients, providing evidence for a single mutation event for each of the three FECH gene mutations in the Swiss population. In addition, two conserved areas, telomeric of the insT213 allele and centromeric of the delTACAG580-584 allele, were identified by analyses of extragenic polymorphisms covering a genomic region of 6.6 cM flanking the FECH gene. No conserved microsatellite haplotypes were found to be associated with any of the three mutations, however. Although these results do not allow an accurate calculation of the age of the mutations, it is reasonable to conclude that these recurrent FECH gene mutations have been present in the Swiss EPP population for a relatively long time. Recurrent mutations or “founder effects” have been observed in other disease-associated populations in Switzerland. Molecular studies of the porphobilinogen deaminase gene unveiled a common mutation of W283X, with a prevalence of around 60%, among Swiss patients with acute intermittent porphyria (Schneider-Yin et al., 2000bSchneider-Yin X. Bogard C. Rüfenacht U. Puy H. Nordmann Y. Minder E.I. Deybach J. Identification of a prevalent nonsense mutation (W283X) and two novel mutations in the porphobilinogen deaminase gene of Swiss patients with acute intermittent porphyria.Hum Hered. 2000; 50: 247-250Crossref PubMed Scopus (21) Google Scholar;Schuurmans et al, in press). A further example for a recent founder effect in the Swiss population is the high incidence (approximately 5%) of the cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation insT3905 among patients with cystic fibrosis, as evidenced by its association with a conserved haplotype of three highly polymorphic intragenic microsatellites (Hergersberg et al., 1997Hergersberg M. Balakrishnan J. Bettecken T. et al.A new mutation, 3905insT, accounts for 4.8% of 1173 CF chromosomes in Switzerland and causes a severe phenotype.Hum Genet. 1997; 100: 220-223https://doi.org/10.1007/s004390050494Crossref PubMed Scopus (11) Google Scholar). It is therefore tempting to speculate that the Swiss population is at least partly constituted from relatively homogeneous subpopulations. As shown in this study, the utilization of intragenic SNP in the FECH gene played a decisive role in the search for ancestral founders of various mutations. Our results supported the view that SNP, due to their low mutation rates, are appropriate markers for the identification of founder haplotypes. Using extragenic microsatellite markers, Wang and colleagues studied haplotypes of EPP patients from unrelated families carrying identical FECH gene mutations commonly found in the U.S.A. (Wang et al., 1999Wang X. Yang L. Kurtz L. et al.Haplotype analysis of families with erythropoietic protoporphyria and novel mutation of the ferrochelatase gene.J Invest Dermatol. 1999; 113: 87-92Crossref PubMed Scopus (18) Google Scholar). The questions raised from their study as to whether the two common mutations were “hotspots” or “dispersed ancestral mutant alleles” in the FECH gene might be clarified by the analysis of the intragenic SNP haplotypes. The hereditary mode of EPP has long been a discussion point. The description “an autosomal dominant disorder with a low clinical penetrance” has been the most commonly used term in EPP, although in some respects the disease resembles an autosomal recessive disorder. Based on clinical and biochemical analyses of over 200 EPP patients, Went and Klasen proposed a “third allele” hypothesis to explain the low clinical penetrance in EPP (Went and Klasen, 1984Went L. Klasen E. Genetic aspects of erythropoietic protoporphyria.Ann Hum Genet. 1984; 48: 105-117Crossref PubMed Scopus (111) Google Scholar). In this hypothesis, an unknown factor or a “third allele” in addition to the defective enzyme was postulated to be responsible for the clinical manifestation of EPP. The hypothetical “third allele” was substantiated through the identification of a low expressed FECH allele in a number of EPP families (Gouya et al., 1996Gouya L. Deybach J.C. Lamoril J. et al.Modulation of the phenotype in dominant erythropoietic protoporphyria by low expression of the normal ferrochelatase gene.Am J Hum Genet. 1996; 58: 292-299PubMed Google Scholar,Gouya et al., 1999Gouya L. Puy H. Lamoril J. et al.Inheritance in erythropoietic protoporphyria: a common wild-type ferrochelatase allelic variant with low expression accounts for clinical manifestation.Blood. 1999; 93: 2105-2110PubMed Google Scholar). Hence, the full expression of EPP phenotype requires the coinheritance of a “normal” FECH allele that has low expression and a mutant FECH allele (Gouya et al., 1999Gouya L. Puy H. Lamoril J. et al.Inheritance in erythropoietic protoporphyria: a common wild-type ferrochelatase allelic variant with low expression accounts for clinical manifestation.Blood. 1999; 93: 2105-2110PubMed Google Scholar). The extensive genetic information gathered in this study enabled us to verify Gouya's latest findings. As seen in all pedigrees, a disease-precipitating nonmutant allele cosegregated with the mutant FECH allele in patients. These nonmutant, presumably low expressed, alleles were characterized by an SNP haplotype [-251G; IVS1–23T] in six out of seven families. With the exception of the two SNP, no other common features, either intragenic or extragenic, were observed among the nonmutant alleles in all seven EPP pedigrees. Undoubtedly, the [-251G; IVS1–23T] haplotype is strongly linked to the low expressed FECH allele, as Gouya et al concluded in their studies, as the same haplotype was identified in the low expressed normal FECH alleles from the six French EPP families tested (Gouya et al., 1996Gouya L. Deybach J.C. Lamoril J. et al.Modulation of the phenotype in dominant erythropoietic protoporphyria by low expression of the normal ferrochelatase gene.Am J Hum Genet. 1996; 58: 292-299PubMed Google Scholar,Gouya et al., 1999Gouya L. Puy H. Lamoril J. et al.Inheritance in erythropoietic protoporphyria: a common wild-type ferrochelatase allelic variant with low expression accounts for clinical manifestation.Blood. 1999; 93: 2105-2110PubMed Google Scholar). We did find an exception of a nonmutant FECH allele carrying [-251 A; IVS1–23C] in one pedigree (family VII), however. Interestingly, although the index patient in this family suffers from typical clinical symptoms of EPP, the mother of the index patient, now aged 74, used to be symptomatic until the age of 35. Except for the fact that the low expressed FECH allele has a reduced mRNA output, little is known about the mechanism of low expression. The role of SNP -251 A/G on the transcriptional activity of FECH was recently examined byMagness et al., 2000Magness S. Tugores A. Brenner D. Analysis of ferrochelatase expression during hematopoietic development of embryonic stem cells.Blood. 2000; 95: 3568-3577PubMed Google Scholar. Two FECH promoters of 1.1 kb, one with an A and the other with a G at position -251, were cloned separately in front of a reporter gene. No differences in terms of the amount of protein generated by the reporter gene were observed between the two promoters. The C→T transition at position -23 in intron 1 can indeed cause the skipping of exon 2 as observed byNakahashi et al., 1992Nakahashi Y. Fujita H. Taketani S. Ishida N. Kappas A. Sassa S. The molecular defect of ferrochelatase in a patient with erythropoietic protoporphyria.Proc Natl Acad Sci USA. 1992; 89: 281-285Crossref PubMed Scopus (81) Google Scholar. IVS1–23T, however, by way of exon 2 skipping, is certainly not the only explanation for the low mRNA output as, at least in family VII, the EPP patient was homozygous C/C at IVS1-23. The continuing search for a functional mutation in the low expressed FECH allele may eventually lead to the clarification of the low expression mechanism in EPP. The authors wish to thank Dr. David Betts for his help in the preparation of this manuscript. This work was supported by the Swiss National Science Foundation, grant 31-53799.98.
Referência(s)