Abstract Pseudoxanthoma elasticum (PXE) is a herita-ble elastic tissue disorder recently shown to be attribut-able to mutations in the ABCC6 (MRP6) gene. WhereasPXE has been identified in all ethnic groups studied todate, the prevalence of this disease in various populationsis uncertain, although often assumed to be similar. A no-table exception however is the prevalence of PXE amongSouth African Afrikaners. A previous report has sug-
gested that a founder effect may explain the higher preva-lence of PXE in Afrikaners, a European-derived popula-tion that first settled in South Africa in the 17th century.To investigate this hypothesis, we performed haplotypeand mutational analysis of DNA from 24 South Africanfamilies of Afrikaner, British and Indian descent. Amongthe 17 Afrikaner families studied, three common haplo-types and six different disease-causing variants were iden-tified. Three of these mutant alleles were missense vari-ants, two were nonsense mutations and one was a singlebase-pair insertion. The most common variant accountedfor 53% of the PXE alleles, whereas other mutant allelesappeared at lower frequencies ranging from 3% to 12%.Haplotype analysis of the Afrikaner families showed thatthe three most frequent mutations were identical-by-de-scent, indicating a founder origin of PXE in this popula-tion.
Introduction
Pseudoxanthoma elasticum (PXE, MIM 177850, MIM264800) is a heritable connective tissue disorder charac-terized by the accumulation of morphologically abnormaland mineralized elastic fibres in dermal, cardiovascularand ocular tissues (Uitto and Shamban 1987). The dermalphenotype is the most prevalent characteristic of PXE andis frequently associated with dramatic ocular and vascularsymptoms (Nishida et al. 1990; Lebwohl et al. 1993;Weenink et al. 1996). Abnormally calcified elastic fibresaccumulate in the mid-dermis typically producing yellow-ish papules associated with laxity and loss of elasticityand are mainly located within flexural areas particularlythe neck, axilla, antecubital fossa and groin (Uitto andShamban 1987; Neldner 1988; Uitto et al. 1998). Similararterial changes within the internal elastic lamina fre-quently cause premature peripheral vascular occlusivedisease (Nishida et al. 1990; Lebwohl et al. 1993). An-gioid streaks, the other hallmark of PXE, result from thefragmentation and calcification of elastic fibres withinBruch’s membrane. These changes in this elastic mem-
Olivier Le Saux · Konstanze Beck · Christine Sachsinger ·Carina Treiber · Harald H. H. Göring · Katie Curry ·Eric W. Johnson · Lionel Bercovitch · Anna-Susan Marais ·Sharon F. Terry · Denis L. Viljoen · Charles D. Boyd
Evidence for a founder effect for pseudoxanthoma elasticum in the Afrikaner population of South Africa
Received: 17 April 2002 / Accepted: 8 July 2002 / Published online: 7 September 2002
ORIGINAL INVESTIGATION
Electronic-database information: accession numbers and URLsfor data in this article are as follows:Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for PXE [MIM 177850, MIM 264800])Genbank (for BAC clone CIT987SK-A-962B4 [accession numberU91318], for ABCC6 cDNA [accession number NM_001171], forPPOX cDNA [accession number U26446], for FANCC cDNA[accession number XM_047190], for FANCA cDNA [accessionnumber NM_000135])The mutations reported here have been submitted to the HumanGene Mutation Database (HGMD, http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.html), temporary accession number H972168
O.Le Saux · K. Beck · C. Sachsinger · C. Treiber · C.D. Boyd (✉)Pacific Biomedical Research Center, University of Hawai’i, Honolulu, Hawaii, USAe-mail: [email protected], Tel.: +1-808-9566341, Fax: +1-808-9569481
H.H.H. GöringDepartment of Genetics, Southwest Foundation for Biomedical Research, San Antonio, Tex., USA
K. Curry · E.W. JohnsonBarrow Neurological Institute, Phoenix, Ariz., USA
L. BercovitchDepartment of Dermatology, Brown University, Providence, R.I., USA
A.-S. MaraisPXE International (South Africa), Mowbray, South Africa
S.F. TerryPXE International, Sharon, Mass., USA
D.L. ViljoenDepartment of Human Genetics, University of Witwatersrand, Johannesburg, South Africa
brane result in subretinal neovascularization, haemor-rhage and, in many cases, the severe loss of central vision(Weenink et al. 1996).
The mutational analysis of several candidate genes at16p13.1 (Le Saux et al. 1999; Cai et al. 2000) recently re-vealed mutations responsible for PXE in a gene encodingthe ATP-binding cassette sub-family C, member 6 (ABCC6),also known as multidrug-resistance associated protein 6(Bergen et al. 2000; Germain et al. 2000; Le Saux et al.2000; Ringpfeil et al. 2000; Struk et al. 2000). To date,more than 50 mutations, mostly clustered in a region ofthe gene encoding the carboxy-terminal end of ABCC6,have been characterized (Le Saux et al. 2001; Meloni etal. 2001; Pulkkinen et al. 2001).
The genetics and disease characteristics of PXE havebeen largely studied to date in populations of Caucasianorigin. The prevalence of PXE among the Caucasians hasbeen reported to be approximately 1:100,000 live births(Neldner 1988). A similar prevalence of PXE has beennoted in Japan (Katagiri 1991). In a preliminary study,Viljoen (1991) found considerable variation of the preva-lence of this disease between different racial groups inSouth Africa. Torrington and Viljoen (1991) and Viljoen(1991) notably reported a prevalence of 1/650,000 in theblack population in the Cape Province of South Africaand a much higher prevalence of 1/23,000 in the Afrikanercommunity of the Cape Province.
The Afrikaner population is of Dutch, German andFrench Huguenot descent and has its origins in the firstEuropean immigrant settlements at the Cape of GoodHope during the 17th century (Viljoen 1991). Torringtonand Viljoen (1991) have proposed that the basis for thehigh prevalence of PXE in the Afrikaner population is afounder effect, similar to that of other rare disorders thatappear at higher frequencies in this population (Botha andBeighton 1983). An initial genealogical study traced theancestry of 20 present-day families with PXE back to po-tentially only four individuals, suggesting that this disor-der is most likely derived from these original founders inSouth Africa (Torrington and Viljoen 1991). To study thispossibility further, we have carried out haplotype and mu-tational analyses in 17 of the 20 Afrikaner families origi-nally analysed.
Subjects and methods
Patients
The DNA of individuals examined in this study was collected inSouth Africa by D.L.V., A.S.M., S.T. and L.B. on behalf of PXEInternational. The diagnosis of PXE in all unrelated individualswas consistent with previously reported consensus criteria (Chris-tiano et al. 1992). The affected status of an individual was basedupon the presence of dermal lesions, ocular symptoms and cardio-vascular findings and was confirmed by positive von Kossa stain-ing of a skin biopsy. Blood samples were obtained from affectedand unaffected individuals who provided informed consent. Thecollection of blood following informed consent was carried out infull compliance with all established NIH guidelines. GenomicDNA was isolated from EDTA-treated whole blood and purifiedwith a Gentra DNA isolation kit (Gentra Systems, Minneapolis,
Minn.) according to the manufacturer’s instructions. Purified DNAwas stored at –80°C in 1 mM TRIS-HCl, 0.1 mM EDTA, pH 8.0.
Polymerase chain reaction and DNA sequence analysis
All 31 exons of ABCC6 were polymerase chain reaction (PCR)-amplified from DNA obtained from the PXE individuals and froma control DNA panel obtained from 54 unaffected and unrelatedAfrikaners. Primers for PCR amplification for all ABCC6 exonswere synthesized based on sequence information obtained frombacterial artificial chromosome (BAC) clone CIT987SK-A-962B4(Genbank accession number U91318; Le Saux et al. 2001). Typi-cal PCR reactions have been described elsewhere (Le Saux et al.1999). PCR products, typically 150–550 bp in length (includingcomplete intron/exon boundaries), were analysed either by single-strand conformation polymorphism analysis (Orita et al. 1989), byconformation-sensitive gel electrophoresis (Ganguly et al. 1993)or by direct sequencing of ABCC6 coding regions. The criteria fordefining whether an allele was disease-causing or a neutral orsilent variant were as previously described (Le Saux et al. 2001).DNA sequence analysis was performed by using ABI BigDye ter-minator cycle sequencing with an ABI310 automated DNA se-quencer.
Haplotype analysis
Haplotype analysis was performed by using previously describedmethods and a set of microsatellite markers spanning region p13.1of chromosome 16 (Le Saux et al. 1999, 2001). Radiolabelled PCRproducts were analysed by autoradiography following elec-trophoresis on 6% polyacrylamide gels containing 6 M urea,37.5% formamide and TBE buffer (0.09 M TRIS, 0.09 M boricacid, 0.01 M EDTA pH 8.0). Electrophoresis was conducted at 80 W for 4–6 h and gels were then dried and exposed to autoradi-ographic film at –80°C for 8-72 h. The genotypes of the variousfamily members were determined “blind” without prior informa-tion describing individual affectation status. Analysis of haplo-types was performed both manually and by using the haplotypesub-routine of the Cyrillic software package (Cherwell Scientific,Buckinghamshire, UK).
Results
Identification ABCC6 gene mutations in Afrikaner PXE patients
To identify the Afrikaner PXE mutations, we screened all31 exons of the ABCC6 gene in DNA samples from 17 ofthe 20 Afrikaner PXE patients and their family memberspreviously examined by Torrington and Viljoen (1991).We also analysed DNA samples from seven other SouthAfrican PXE patients and their family members. ThesePXE families were of black South African, Indian andBritish ancestries.
Among the Afrikaners, six different mutant alleleswere detected (Table 1). One missense allele, R1339C,represented more than half (53%) the total number ofAfrikaner mutant alleles (Table 2). Other ABCC6 mutantalleles included two missense mutations, R1138Q andL673P, which occurred at frequencies of 8.8% (3 out of 34 alleles) and 2.9% (1 out of 34 alleles), respectively.Two nonsense variants (Y768X and R1141X) and a singleframeshift mutation (939insT) were also identified (Table 1). The latter mutant alleles occurred at frequencies
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of 11.8% (4 out of 34 alleles), 5.9% (2 out of 34 alleles)and 2.9% (1 out of 34 alleles), respectively (Table 2). Twoof the mutations found in the Afrikaner group, R1141Xand 939insT, were also found in PXE individuals ofBritish ancestries (Tables 1, 2). One of the latter individu-als (from Family 212) carried the R1141X allele in asso-ciation with 939insT. The two 939insT alleles found inAfrikaner and British PXE individuals were most proba-bly identical-by-descent as they occurred in a conserved
haplotype shared by Family 212 (British ancestry) andFamily 213 (Afrikaner), as discussed below. Moreover,two of the four R1141X alleles found in Afrikaner andBritish individuals occurred in similar haplotypes, alsosuggesting a possible consanguineous relationship be-tween Family 206 (Afrikaner) and Family 216 (British an-cestry). The remaining R1141X mutations, characterizedin Family 224 (Afrikaner) and Family 212 (British ances-try) were found in different haplotypes indicating that
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Table 1 ABCC6 mutations identified in a cohort of South African PXE patients of Afrikaner and other ancestries (nt nucleotide, aa amino acid, ni not identified, UK United Kingdom, Black black South Africans)
Family Allele 1 Allele 2 Ancestry
nt change aa change Exon Haplotype nt change aa change Exon Haplotype
201 4015C→T R1339C 28 I 4015C→T R1339C 28 I Afrikaner203 4015C→T R1339C 28 I 3413G→A R1138Q 24 III Afrikaner205 3413G→A R1138Q 24 III 2304C→A Y768X 18 II Afrikaner206 4015C→T R1339C 28 I 3421C→T R1141X 24 Other Afrikaner208 4015C→T R1339C 28 I 4015C→T R1339C 28 I Afrikaner209 4015C→T R1339C 28 I 2018T→C L673P 16 Other Afrikaner211 4015C→T R1339C 28 I 3413G→A R1138Q 24 III Afrikaner222 4015C→T R1339C 28 I 4015C→T R1339C 28 I Afrikaner223 4015C→T R1339C 28 I 2304C→A Y768X 18 II Afrikaner225 4015C→T R1339C 28 I 4015C→T R1339C 28 I Afrikaner226 4015C→T R1339C 28 I 2304C→A Y768X 18 II Afrikaner228 4015C→T R1339C 28 I 2304C→A Y768X 18 II Afrikaner229 4015C→T R1339C 28 I 4015C→T R1339C 28 I Afrikaner213 939insT Frameshift 8 Other ni ni ni Other Afrikaner214 4015C→T R1339C 28 I ni ni ni Other Afrikaner224 3421C→T R1141X 24 Other ni ni ni Other Afrikaner204 ni ni Other ni ni ni Other Afrikaner212 3421C→T R1141X 24 Other 939insT Frameshift 8 Other UK215 3775delT Frameshift 27 Other 4104delC Frameshift 29 Other UK217 ABCC6del15 Frameshift 15 Other ABCC6del15 Frameshift 15 Other Indian207 3088C→T R1030X 23 Other ni ni ni Other UK216 3421C→T R1141X 24 Other ni ni ni Other UK219 1553G→A R518Q 12 Other ni ni ni Other UK221 ni ni Other ni ni ni Other Black
Table 2 Frequencies of mu-tant ABCC6 alleles found in acohort of PXE patients ofAfrikaner and other ancestries(? unidentified alleles, MDRmutation detection rate)
these alleles were either of independent origin or that theirco-ancestry was obscured by historical recombination andmutation events (Le Saux et al. 2001). Several other mu-tant alleles in compound heterozygous or homozygousstates were characterized in the seven non-Afrikaner indi-viduals examined (Table 1). Some of these mutant alleleshad been previously identified in other patient cohorts (LeSaux et al. 2001; Ringpfeil et al. 2001) and a homozygousABCC6del15 allele was found in a single consanguineousfamily of Indian origin (Table 1). This AluSx mediateddeletion encompassed 1583 nucleotides from IVS14+1530to IVS15+949 deleting all of exon 15, and corresponded
to an out-of-frame deletion creating a premature stop-codonat position 624 (Le Saux et al. 2001).
Haplotype analysis of Afrikaner pedigrees
Thirteen different haplotypes were characterized in PXEpatients from the 17 Afrikaner families that we examinedby mutation analysis. Three major haplotypes, with somevariation at single markers, encompassing the PXE locus(located between markers D16S405 and D16S764; LeSaux et al. 1999; Cai et al. 2000) were identified (Fig. 1)and three disease alleles (R1339C, Y768X, R1138Q) werefound to be strictly associated with these three haplotypes(referred to as types I, II and III; Fig.1).
Haplotypes I, II and III were found either in combina-tion or in a homozygous state in 14 of the 17 PXE probands.One of the haplotypes (type I), which corresponded to astretch of 10 consecutive markers (D16S680 to D16S3017),was found in 13 of these apparently unrelated PXE indi-viduals. Five affected individuals presented this type Ihaplotype in a homozygous state and eight individuals
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Fig.1 Haplotype analysis of a cohort of Afrikaner individualswith PXE. Haplotype studies were performed with 17 D16S poly-morphic markers spanning approximately 25 cM of 16p13.1.Three haplotypes (types I, II, III) are presented together with theassociated mutant alleles. Afrikaner families are numbered201–229. Markers indicated in bold define the conserved regions(boxed) that are likely to be derived from the three founderAfrikaner individuals. Underlined numbers Divergent markerswithin the conserved haplotype regions, asterisk an ambiguousgenotype
were heterozygous. The other two haplotypes (types IIand III), which corresponded to stretches of 12 and 11consecutive markers (D16S680 to D16S417 and D16S497to D16S3103, respectively), were only found in heterozy-gous states in three and four unrelated individuals, respec-tively (Fig.1). Some affected individuals with haplotypestype I and III shared five non-consecutive markers distalto the PXE locus (from D16S497 to D16S3127) suggest-ing that recombination occurred between these chromo-somes. The three conserved haplotypes (types I, II and III)were not found in any of the DNA samples obtained fromindividuals of British, Indian or black South African an-cestries.
A family of Afrikaner origin (Family 213) and a fam-ily of British ancestry (Family 212) both displayed a com-mon haplotype at seven consecutive markers telomeric tothe PXE locus (D16S407 to D16S3127) and presentedseveral other alleles in common with two of the three hap-lotypes. This region was most probably inherited identi-cal-by-descent. This shared haplotype between both fami-lies suggests some cross-cultural admixture between Eng-lish and Afrikaner settlers and indeed, such inter-mar-riages were not uncommon in the later evolution of SouthAfrican European-derived communities in the 19th and20th centuries (Beighton 1976; Viljoen 1991).
The three disease alleles, R1339C, Y768X, and R1138X,associated with the type I, II and III haplotypes representthree ancestral founder alleles from which 74% of the
Afrikaner alleles have descended. It is highly likely thatthese three alleles were introduced into the Afrikaner pop-ulation from founder settlers.
All other mutated alleles, R1141X, L673P, 939insTand particularly the five unidentified mutations, werefound in divergent haplotypes. The latter three alleles(R1141X, L673P and 939insT) could have been intro-duced by founder individuals or could represent de novomutation events in the Afrikaner population. Alterna-tively, these mutations could have been introduced intothe Afrikaner gene pool through cross-cultural marriageswith other populations groups.
No ABCC6 mutations were found in an Afrikaner fam-ily (Family 204) and a family of South African black an-cestry (Family 221). Although it is possible that PXE inthese families could be attributable to mutations in a geneother than the ABBC6 gene, to date all reported linkageand mutational studies have demonstrated that the PXEphenotype develops as a consequence of ABCC6 muta-tions (Le Saux et al. 2001). It is probable therefore thatmutations in the ABCC6 gene that we have not yet de-tected will be responsible for PXE in these two familiesand, moreover, that the second alleles that have not yetbeen identified in other, apparently heterozygous, PXEpatients will also be mutations in the ABCC6 gene.
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Table 3 Silent and neutralvariants identified in theABCC6 gene in a cohort of 24South African patients withPXE (hm homozygote, ht het-erozygote, aa amino acidchanges, nt nucleotide changes,i- the intron in which the vari-ant is located, No. of allelesnumber of variants found inthe 48 PXE chromosomesanalysed in this study
aVariants identified by se-quencing only; variants identi-fied in either or both ABCC6pseudogenes have not been in-dicated in this table
aa nt Status Exon Origin No. of alleles
G61D 182G→A ht 2 Afrikaner 1T215T 645G→A ht 6 Afrikaner 2K281Ea 841A→G ht, hm 8 Afrikaner, UK 2T285Ta 855C→T ht, hm 8 Afrikaner, UK 2I319Va 955A→G ht, hm 8 Afrikaner, UK 2N411N 1233T→C ht, hm 10 Afrikaner, UK 20V415V 1245G→A ht, hm 10 All 20none IVS11+73G→C ht i-11 Afrikaner, UK 2none IVS11-45C→A ht i-11 UK 1none IVS11-41A→G ht, hm i-11 Afrikaner, UK 5none IVS11-22C→A ht i-11 Afrikaner 1V614A 1841T→C ht, hm 14 All 16T630Ta 1890C→G ht, hm 15 Afrikaner, UK 11H632Qa 1896C→A ht, hm 15 Afrikaner, UK 11A830A 2490C→T ht, hm 19 Afrikaner, UK 6none IVS21+30G→A ht i-21 UK 2P945P 2835C→T ht, hm 22 Indian, UK 3none IVS22-5delTCCC-8 ht i-22 UK 1none IVS24-16T→C ht i-24 UK 1none IVS24-3C→T ht i-24 AFK 1none IVS25+55T→C ht i-25 Afrikaner, UK 8none IVS25+90G→A ht, hm i-25 All 21R1268Q 3803G→A ht, hm 27 Afrikaner, UK 4none IVS27-6G→A ht i-27 Afrikaner 1none IVS28+49C→T ht, hm i-28 Afrikaner 18I1350L 4048A→C ht 29 UK 1none 3’ UTR+17G→A ht 3’UTR Afrikaner 2
Silent and neutral variants of ABCC6
An additional 27 variants, assumed to be neutral or silent,were identified in the course of the ABCC6 screening ofour cohort of 24 South African PXE individuals (Table 3).These variants did not reveal any clustering or an evidentethnic segregation. Of these nucleotide changes, 21 wereprobably silent variants (unlikely to influence the pheno-type) as they occurred in either non-coding regions (14variants) or did not modify the encoded amino acid. Sixother variants (G61D, K281E, I319V, V614A, H632Q andR1268Q) resulted in amino acid changes but appeared notto segregate with the disease in PXE pedigrees and weretherefore likely to be neutral. Although variant G61D rep-resents a significant amino acid change, this allele wasfound in an Afrikaner patient homozygous for the muta-tion R1339C and was therefore considered to be a neutralvariant. It is noteworthy that one silent variant (IVS28+49C→T) co-segregated with the R1339C alleles, as it wasfound in very close proximity (76 bp) to the 4015C→Tsubstitution responsible for the arginyl to cysteinyl change(Tables 1, 3). No variants were found to co-segregate withthe two other founder alleles R1138Q and Y768X.
The prevalence of PXE in the Afrikaner population
In our previous studies (Le Saux et al. 2000, 2001), ouroverall criteria for defining a disease allele included thepre-requisite that such an allele would not be present incontrol chromosomes derived from unaffected individu-als. As the occasional presence of disease alleles in a het-erozygous state among unaffected individuals is verylikely in the largely consanguineous Afrikaner population,this rule cannot be applied in the present study. Indeed,upon screening the ABCC6 gene in the 54 unaffectedAfrikaner controls used in this study, two mutant alleleswere identified. R1139C and an out-of-frame 25-bp dele-tion (865-889del) were found in a heterozygous state intwo apparently unrelated individuals. Although 865-889delwas not identified in the 34 chromosomes analysed inAfrikaner PXE patients, data from the present study andprevious results (Le Saux et al. 2001) indicated thatR1339C is disease-causing and represents the most preva-lent founder mutation found to date among South AfricanAfrikaners.
From the presence of a single R1339C allele in ourcontrol panel of 54 apparently unrelated and unaffectedAfrikaners, we cannot calculate an accurate estimate ofprevalence of these founder mutations in the Afrikanerpopulation, as a much larger cohort of Afrikaners willneed to be screened. It is encouraging however that, inonly 108 chromosomes, a founder mutant was observed.This suggests that the prevalence of PXE, propagated byat least the three founder alleles that we have identified todate in the South African Afrikaners, will indeed be rela-tively high.
Discussion
The present study describes the identification of threePXE disease alleles, R1339C, Y768X and R1138Q, whichare associated with three conserved haplotypes (types I, IIand III) and which account for 74% of the PXE disease al-leles among South African Afrikaners. These alleles arenot found in patients of other ancestries in South Africa.Our findings suggest that at least 14 of the 17 AfrikanerPXE patients were derived from three common ancestorsand these results are consistent with a founder effect in theAfrikaner population of South Africa with PXE as origi-nally proposed by Torrington and Viljoen (1991). How-ever, the authors of the original founder hypothesis wereunable clearly to establish independent lineages betweenfour suspected founder individuals (Torrington andViljoen 1991; Viljoen 1991). The present results suggestthat at least three separate founder individuals carryingmutant ABCC6 alleles settled in the area of the CapeProvince. It is probable that these European-derived indi-viduals brought these mutant alleles into the emergingAfrikaner population (and represent therefore true founders),although it is possible that any one of these mutationscould also have been created de novo in the Afrikanercommunity. Identification of these mutant alleles withinthe appropriate haplotypes in ancestral European commu-nities should confirm on which continent these allelesoriginated.
There are multiple examples of heritable recessive dis-orders with unusually high prevalence in the Afrikaners ofSouth Africa, most (if not all) of which are the result offounder effects. Familial hypercholesterolaemia is themost extensively studied with a prevalence of 1/80 amongthe Afrikaans-speaking population in South Africa, com-pared with 1/500 in other communities (Jooste et al. 1986;Steyn et al. 1996). Three founder mutations, D206E,V408M, and D154N, have been identified in the low-den-sity lipoprotein receptor gene (Leitersdorf et al. 1989;Kotze et al. 1989, 1991) and represent 90% of all muta-tions detected in this gene within this Afrikaner cohort.Another example of a founder effect that results in an in-creased prevalence of a disorder in Afrikaners is por-phyria variegata. More than 95% of porphyria families ofAfrikaner origin carry an R59W variant of the PPOX gene(Meissner et al. 1996). Fanconi anaemia, a rare autosomalrecessive disorder, also has a high prevalence in Ashke-nazi Jews and Afrikaners. Among Ashkenazi Jews, mostcases of Fanconi anaemia are caused by a single splicesite mutation in the FANCC gene (Whitney et al. 1993).Among South African Afrikaners, a recent molecular andgenealogical study has revealed a strict association be-tween mutations in the FANCA gene and conserved haplo-types on chromosome 16, with the most frequent mutationin the FANCA gene accounting for 60% of the disease al-leles (Tipping et al. 2001).
As a heritable disease, PXE demonstrates a high de-gree of variable expression. Earlier literature has sug-gested, for example, multiple clinical types of PXE, in-
336
cluding a clinically distinct form of PXE in patients ofAfrikaner origin (Viljoen et al. 1987). Both dominant andrecessive forms of PXE have also been reported, al-though, to date, no dominant forms of PXE have beenconfirmed to segregate with known ABCC6 mutations.Whereas the mutational data accumulated to date suggeststhat most (if not all) cases of PXE are attributable to mu-tations within the ABCC6 gene, it is not yet clear whetherdifferent ABCC6 mutations contribute to the variable ex-pression of the PXE phenotype. Moreover, it is also un-clear (but highly likely) whether the penetrance of ABCC6mutations may be influenced by epigenetic or environ-mental factors (Uitto et al. 2001). Some preliminary evi-dence, for example, suggests that calcium intake in child-hood may be a factor influencing phenotypic severity inadult PXE patients (Renie et al. 1984).
The identification of founder ABCC6 mutations withinthe Afrikaner population should afford diagnostic oppor-tunities that will assist in the treatment of PXE within apopulation in which the disease is far more prevalent thanother population groups studied to date. The high fre-quency of a small number of founder ABCC6 mutationswithin a relatively homogenous population (Gordon et al.2000) should also provide the opportunity, within anAfrikaner cohort, to dissect out the influence of variousABCC6 mutations on penetrance and/or expression of thePXE phenotype. The Afrikaner PXE population may alsoafford the opportunity for studying the epigenetic influ-ences on a select number of ABCC6 mutant alleles; thesecould be crucial not only for the treatment of PXE withinSouth Africa but also for PXE patients in other communi-ties.
Acknowledgements We are very grateful to all affected individ-uals and their relatives for their cooperation in making this studypossible. We also thank the other members of the PXE Interna-tional Research Consortium, particularly Patrick Terry. This workwas supported by NIH grants EY13019 and RR16453 to C.B. andan RCMI (Research Centers in Minority Institutions) grant fromNCRR (RR03061) to the Pacific Biomedical Research Center ofthe University of Hawaii.
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