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Linkage Disequilibrium Analysis in a Recently Founded Population: Evaluation of the Variegate Porphyria Founder in South African Afrikaners

1998; Elsevier BV; Volume: 62; Issue: 5 Linguagem: Inglês

10.1086/301833

1537-6605

J.Z. Groenewald, Junita Liebenberg, Ilse M. Groenewald, Louise Warnich,

Folate and B Vitamins Research

To the Editor: Variegate porphyria (VP; MIM 176200) is relatively rare in most populations, but it is one of the most common autosomal dominant genetic disorders in South Africa (Dean, 1971Dean G The porphyrias: a story of inheritance and environment. 2d ed. Pitman Medical, London1971Google Scholar). The disease is characterized by a diversity of symptoms, including a variable picture of skin symptoms and acute attacks. By means of genealogical studies, the history of VP in South Africa can be traced back to the marriage of a Dutch couple in the Cape of Good Hope in 1688 (Dean, 1971Dean G The porphyrias: a story of inheritance and environment. 2d ed. Pitman Medical, London1971Google Scholar). This, along with the high prevalence of VP in South Africa, has promoted the founder-gene hypothesis for VP in this country. Mutations in the protoporphyrinogen oxidase gene (PPOX), the seventh enzyme in the heme biosynthetic pathway, have been shown to be causative of VP (Deybach et al., 1996Deybach J-C Puy H Robréau A-M Lamoril J Da Silva V Grandchamp B Nordmann Y Mutations in the protoporphyrinogen oxidase gene in patients with variegate porphyria.Hum Mol Genet. 1996; 5: 407-410Crossref PubMed Scopus (49) Google Scholar; Meissner et al., 1996Meissner PN Dailey TA Hift RJ Ziman M Corrigall AV Roberts AG Meissner DM et al.A R59W mutation in human protoporphyrinogen oxidase results in decreased enzyme activity and is prevalent in South Africans with variegate porphyria.Nat Genet. 1996; 13: 95-97Crossref PubMed Scopus (171) Google Scholar; Warnich et al., 1996Warnich L Kotze MJ Groenewald IM Groenewald JZ van Brakel MG van Heerden CJ de Villiers JP et al.Identification of three mutations and associated haplotypes in the protoporphyrinogen oxidase gene in South African families with variegate porphyria.Hum Mol Genet. 1996; 5: 981-984Crossref PubMed Scopus (73) Google Scholar;Lam et al., 1997Lam HM Dragan L Tsou HC Merk H Peacocke M Goerz G Sassa S et al.Molecular basis of variegate porphyria: a de novo insertion mutation in the protoporphyrinogen oxidase gene.Hum Genet. 1997; 99: 126-129Crossref PubMed Scopus (17) Google Scholar). This gene has been mapped to chromosome 1q22 by FISH (Taketani et al., 1995Taketani S Inazawa J Abe T Furukawa T Kohno H Tokunaga R Nishimura K et al.The human protoporphyrinogen oxidase gene (PPOX): organization and location to chromosome 1.Genomics. 1995; 29: 698-703Crossref PubMed Scopus (74) Google Scholar), and the position has been confirmed by linkage analysis (Roberts et al., 1995Roberts AG Whatley SD Daniels J Holmans P Fenton I Owen MJ Thompson P et al.Partial characterization and assignment of the gene for protoporphyrinogen oxidase and variegate porphyria to human chromosome 1q23.Hum Mol Genet. 1995; 4: 2387-2390Crossref PubMed Scopus (55) Google Scholar). Three mutations have been described in South African VP patients, but one of these, a C→T transition at nucleotide position 452 (R59W), was found in ∼90% of patients (Meissner et al., 1996Meissner PN Dailey TA Hift RJ Ziman M Corrigall AV Roberts AG Meissner DM et al.A R59W mutation in human protoporphyrinogen oxidase results in decreased enzyme activity and is prevalent in South Africans with variegate porphyria.Nat Genet. 1996; 13: 95-97Crossref PubMed Scopus (171) Google Scholar; Warnich et al., 1996Warnich L Kotze MJ Groenewald IM Groenewald JZ van Brakel MG van Heerden CJ de Villiers JP et al.Identification of three mutations and associated haplotypes in the protoporphyrinogen oxidase gene in South African families with variegate porphyria.Hum Mol Genet. 1996; 5: 981-984Crossref PubMed Scopus (73) Google Scholar). This mutation spanned a CpG dinucleotide, and, to exclude the possibility of a recurrent mutation, intragenic haplotype studies were undertaken. Mutation R59W was shown to be associated with one of four potential haplotypes defined by two diallelic polymorphisms in exon 1 (Warnich et al., 1996Warnich L Kotze MJ Groenewald IM Groenewald JZ van Brakel MG van Heerden CJ de Villiers JP et al.Identification of three mutations and associated haplotypes in the protoporphyrinogen oxidase gene in South African families with variegate porphyria.Hum Mol Genet. 1996; 5: 981-984Crossref PubMed Scopus (73) Google Scholar), thus supporting the founder hypothesis. However, this was not totally conclusive evidence, since the alleles associated with the R59W mutation are also the common alleles in the normal population for each of the polymorphisms (L. Warnich, unpublished data). If the high incidence of a genetic disease in a particular population is due to a founder effect, most cases studied should have preserved alleles at closely linked loci, presenting the original founder chromosome (Hästbacka et al., 1992Hästbacka J de la Chapelle A Kaitila I Sistonen P Weaver A Lander E Linkage disequilibrium mapping in isolated founder populations: diastrophic dysplasia in Finland.Nat Genet. 1992; 2: 204-211Crossref PubMed Scopus (435) Google Scholar). In recently founded populations, comparable to the South African Afrikaner population, a conserved area of ∼5–20 cM can be expected (Houwen et al., 1994Houwen RHJ Baharloo S Blankenship K Raeymaekers P Juyn J Sandkuijl LA Freimer NB Genome screening by searching for shared segments: mapping a gene for benign recurrent intrahepatic cholestasis.Nat Genet. 1994; 8: 380-386Crossref PubMed Scopus (266) Google Scholar). In the present study we have used linkage disequilibrium (LD) and haplotype analyses to investigate the single-founder hypothesis for VP in South Africa and to evaluate the use of the Afrikaner population for future LD mapping studies. In the current study, 15 nuclear families with the R59W mutation (Warnich et al. Warnich et al., 1996Warnich L Kotze MJ Groenewald IM Groenewald JZ van Brakel MG van Heerden CJ de Villiers JP et al.Identification of three mutations and associated haplotypes in the protoporphyrinogen oxidase gene in South African families with variegate porphyria.Hum Mol Genet. 1996; 5: 981-984Crossref PubMed Scopus (73) Google Scholar, Warnich et al., 1996Warnich L Meissner PN Hift RJ Louw JH van Heerden CJ Retief AE Mapping of the variegate porphyria (VP) gene: contradictory evidence for linkage between VP and microsatellite markers at chromosome 14q32.Hum Genet. 1996; 97: 690-692Crossref PubMed Scopus (8) Google Scholar) were extended to include 132 members, 58 of whom were affected. The 15 families were unrelated to the second-degree and included one four-generation, seven three-generation, and seven two-generation pedigrees. A sequence-tagged site (STS) at the 3′ end of the PPOX gene was used to screen the CEPH YAC libraries. The primers used were D38537-F (5′-GGG AGT TGC TGT TAA TGA CTG T-3′) and D38537-R (5′-GCA ATT TTT ATT TTC ATG AAT GAG-3′). One of the positive YAC clones, 910_C_8, showed an unambiguous hit for two microsatellite markers, D1S2705 and D1S484. Thirteen other microsatellite markers flanking these markers (listed in table 1) and spanning ∼21 cM, were subsequently selected from the Généthon (http://gdbwww.gdb.org) and Cooperative Human Linkage Center (CHLC; http://www.chlc.org) databases.Table 1Allele Frequencies and LD Results for the Most Common Alleles of the 15 R59W-Mutation Chromosomes and for 88 Normal ChromosomesFrequency of ChromosomeDistance to Next Marker LocusaObtained from the Généthon and CHLC databases and from the sex-averaged map of the Marshfield Medical Research Foundation (http://www.marshmed.org/genetics/). (cM)Marker LocusNormalbIncludes chromosomes from 27 individuals who were relatives of the families by marriage, as well as 34 normal chromosomes of affected parents.DiseasecCalculated by counting, with use of the oldest R59W-mutation chromosome in each family.χ2 (P)dCalculations for statistical significance of data were done for the most common allele of each marker, in a pairwise manner using the χ2 test with 1 df and no correction (Dawson-Saunders and Trapp 1990, pp. 150–151).PexcessD1S2140.34.47.8805 (.3481).1908.7D1S303.60.873.892 (.0485).6648.0D1S1595.18.331.8126 (.1782).18523.0D1S1600.17.539.7301 (.0018).4374.0D1S1653.09.5319.1183 (<.0001).48671.5D1S398.33.532.3122 (.1284).30402.8D1S2707.30.9321.2613 (<.0001).90441.2D1S484.30.9321.9526 (<.0001).90541.2D1S2705.28.9322.9604 (<.0001).9069.0D1S1679.16.6718.4746 (<.0001).60364.8D1S104.18.7320.1534 (<.0001).6741.0D1S1677.38.809.4091 (.0022).68002.2D1S426.10.5317.2799 (<.0001).4802.6ATA38A05.15.4711.7399 (.0006).45243.0D1S196.26 .30.47 .472.6167 (.1057) 1.7253 (.1890).2779 .2430a Obtained from the Généthon and CHLC databases and from the sex-averaged map of the Marshfield Medical Research Foundation (http://www.marshmed.org/genetics/).b Includes chromosomes from 27 individuals who were relatives of the families by marriage, as well as 34 normal chromosomes of affected parents.c Calculated by counting, with use of the oldest R59W-mutation chromosome in each family.d Calculations for statistical significance of data were done for the most common allele of each marker, in a pairwise manner using the χ2 test with 1 df and no correction (Dawson-Saunders and Trapp, 1990Dawson-Saunders B Trapp RG Basic and clinical biostatistics. Prentice Hall, Englewood Heights, NJ1990Google Scholar). Open table in a new tab Haplotypes were constructed in each family under the assumption that there were the minimum number of recombinations. Disease-associated haplotypes were identified from alleles that were transmitted from affected parent to affected offspring, in each pedigree. Crossover events on the disease-associated chromosomes of two different individuals placed the PPOX gene telomeric of marker D1S2707 in one of them and centromeric of marker D1S2705 in the other. These observed recombinations delimit the location of the PPOX gene to a 2.4-cM region between markers D1S2705 and D1S2707, and they thus represent the highest-resolution genetic mapping of the gene yet. LD studies were done by calculation of the statistical factor Pexcess for the dominant disease-associated allele of each marker (Hästbacka et al., 1992Hästbacka J de la Chapelle A Kaitila I Sistonen P Weaver A Lander E Linkage disequilibrium mapping in isolated founder populations: diastrophic dysplasia in Finland.Nat Genet. 1992; 2: 204-211Crossref PubMed Scopus (435) Google Scholar). The number of generations since the introduction of the VP gene was taken, on the basis of available genealogical records, as 12. The data generated are shown in table 1. The strongest association was observed at D1S2707 (allele 3), D1S484 (allele 2), and D1S2705 (allele 4), yielding Pexcess values of .9044, .9054, and .9069, respectively. Two-point linkage analysis (data not shown) also illustrated close linkage of the disease locus to these three markers, with LOD scores of 9.37, 12.68, and 10.74 at recombination fraction (θ) values of .031, .023, and .014, respectively. The extended haplotype associated with the R59W mutation in each family is shown in figure 1. Allele 3 was found to be conserved for marker D1S2707 in all of the families with the R59W mutation, except family B. Alleles 2 and 4 were detected for the markers D1S484 and D1S2705 in all of the families, except family L. Since the latter two markers are the nearest to the gene, we propose that family L most likely has an independent R59W mutation. It could thus be deduced that a small percentage of families with the R59W mutation will represent either recurrent mutations at the CpG hot spot or recent importations of the gene. It is interesting to note that the haplotype of family L could not be distinguished from the haplotypes of the other families when diallelic intragenic markers were used (Warnich et al. Warnich et al., 1996Warnich L Groenewald JZ Groenewald IM Kotze MJ Retief AE Molecular genetic evidence for a founder effect in variegate porphyria in South Africa.Braz J Genet. 1996; : 235Google Scholar, Warnich et al., 1996Warnich L Kotze MJ Groenewald IM Groenewald JZ van Brakel MG van Heerden CJ de Villiers JP et al.Identification of three mutations and associated haplotypes in the protoporphyrinogen oxidase gene in South African families with variegate porphyria.Hum Mol Genet. 1996; 5: 981-984Crossref PubMed Scopus (73) Google Scholar). As shown in figure 1, two distinct subhaplotypes were observed surrounding the core haplotype—namely, the haplotype represented by families A, P, G, M, and D and the haplotype depicted by families B, K, J, E, C, O, N, H, and F. It is thus expected that variations in these subhaplotypes can be ascribed to earlier historical recombination events and/or mutations at some loci. We thus believe that, although there are apparently two groups of haplotypes that differ in flanking markers, they both descend from the same founder, because they share the same core haplotype, as has also been found in other founder-related studies (e.g., Labuda et al., 1996Labuda M Labuda D Korab-Laskowska M Cole DEC Zietkiewicz E Weissenbach J Popowska E et al.Linkage disequilibrium analysis in young populations: pseudo–vitamin D–deficiency rickets and the founder effect in French Canadians.Am J Hum Genet. 1996; 59: 633-643PubMed Google Scholar). There is also no geographical or genealogical evidence for two independent introductions of the VP gene in South Africa. Furthermore, a contiguous area of 10 cM (spanned by markers D1S2707 and ATA38A05) displayed highly significant (P<.005) LD values (table 1). These results are in agreement with data from other populations, in which the historical age of the founder effect was estimated to be 12 (Labuda et al., 1996Labuda M Labuda D Korab-Laskowska M Cole DEC Zietkiewicz E Weissenbach J Popowska E et al.Linkage disequilibrium analysis in young populations: pseudo–vitamin D–deficiency rickets and the founder effect in French Canadians.Am J Hum Genet. 1996; 59: 633-643PubMed Google Scholar), 8–12 (Puffenberger et al., 1994Puffenberger EG Kauffman ER Bolk S Matise TC Washington SS Angrist M Weissenbach J et al.Identity-by-descent and association mapping of a recessive gene for Hirschprung disease on human chromosome 13q22.Hum Mol Genet. 1994; 3: 1217-1225Crossref PubMed Scopus (200) Google Scholar), and 5–12 generations (Houwen et al., 1994Houwen RHJ Baharloo S Blankenship K Raeymaekers P Juyn J Sandkuijl LA Freimer NB Genome screening by searching for shared segments: mapping a gene for benign recurrent intrahepatic cholestasis.Nat Genet. 1994; 8: 380-386Crossref PubMed Scopus (266) Google Scholar). In two other studies based on South African families, a conserved region of ∼8 cM was found in two long-QT families with continuing genealogical studies already extending back through nine generations (de Jager et al., 1996de Jager T Corbett CH Badenhorst CW Brink PA Corfield VA Evidence for a long QT founder gene with varying phenotypic expression in South African families.J Med Genet. 1996; 33: 567-573Crossref PubMed Scopus (41) Google Scholar), whereas an ancestral haplotype of 11 cM was found in 11 of 14 South African families with keratolytic winter erythema (Starfield et al., 1997Starfield M Hennies HC Jung M Jenkins T Wienker T Hull P Spurdle A et al.Localization of the gene causing keratolytic winter erythema to chromosome 8p22-p23, and evidence for a founder effect in South African Afrikaans-speakers.Am J Hum Genet. 1997; 61: 370-378Abstract Full Text PDF PubMed Scopus (28) Google Scholar). Large shared segments are expected around disease genes in recently founded populations such as the Afrikaner population, and it was thus predicted that genome searches for these segments could be performed with only a few hundred markers (Houwen et al., 1994Houwen RHJ Baharloo S Blankenship K Raeymaekers P Juyn J Sandkuijl LA Freimer NB Genome screening by searching for shared segments: mapping a gene for benign recurrent intrahepatic cholestasis.Nat Genet. 1994; 8: 380-386Crossref PubMed Scopus (266) Google Scholar). This potentially powerful approach of LD mapping has, however, not been widely used in the past, one of the reasons being the scarcity of suitable founder populations. The next phase of gene mapping—namely, the mapping of complex traits—may especially benefit from conserved-haplotype detection and LD mapping in isolated populations (Lander and Schork, 1994Lander ES Schork NJ Genetic dissection of complex traits.Science. 1994; 265: 2037-2048Crossref PubMed Scopus (2678) Google Scholar). Although the Afrikaner population is known to have founder effects for a number of genetic disorders (Jenkins, 1990Jenkins T Medical genetics in South Africa.J Med Genet. 1990; 27: 760-779Crossref PubMed Scopus (27) Google Scholar), it has rarely been exploited for the actual mapping of genes in the past. From the results of the present study we conclude that the high frequency of the R59W mutation in South Africa could probably be ascribed to a common ancestor and is not due to multiple mutation events on a common haplotype. The current study thus not only provides the first firm molecular evidence for a founder hypothesis for VP but also shows that the South African Afrikaner population is a valuable candidate population for future mapping studies using LD analyses. We are grateful to Dr. Eric Schoenmakers and C. Huysmans of the Center for Human Genetics, University of Leuven (Leuven, Belgium) for screening the YAC libraries. We also thank Drs. Bruce Weir and Eden Martin for helpful discussions during the course of this work. The work was supported by the South African Medical Research Council, the Harry Crossley Trust, and the University of Stellenbosch.