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AGG interspersion patterns in the CGG repeat of the FMR1 gene and linked DXS548/FRAXAC1 haplotypes in Brazilian populations

We have recently described in this journal the distribution of CGG repeats of the FMR1 gene, the allelic frequencies at DXS548 and FRAXAC1 microsatellite loci, and DXS548/FRAXAC1 haplotype frequencies in African-derived, European-derived, and Amerindian populations from South America [Mingroni-Netto et al., 2002]. The CGG repeat sequences and AGG interspersion patterns were not available at that time. A number of haplotype studies in affected males with the fragile X syndrome pointed to two main pathways for the origin of the fragile X mutation in European-derived populations. The two most frequent DXS548/FRAXAC1 haplotypes linked to the fragile X mutation have been found to be associated with CGG repeat structures in the general population, which would favor expansions: the haplotype 2-1 (204/158 bp) is associated to long repeats with uninterrupted CGG arrays, leading to predisposed chromosomes, which would increase in size gradually; the haplotype 6-4 (196/152 bp) appears associated to smaller repeats particularly prone to expansions because of the loss of AGG interruptions at the 30 end of the repeat, also giving rise to long uninterspersed CGG arrays [Eichler et al., 1996]. In the city of São Paulo, Brazil, we observed that these two haplotypes were the most frequent among fragile X patients [MingroniNetto et al., 1999]. More recently, Crawford et al. [2000a] found that the two most prevalent haplotypes in African-American fragile X patients were 4-4 (200/152 bp) and 3-4 (202/152 bp). In normal African-American populations, the haplotype 4-4 was linked to CGG repeats devoid of AGG interruptions near the 50 end of the repeat [Crawford et al., 2000b]. These findings suggested that the pathways leading to fragile X mutations in Africans might be distinct from those leading to mutations in Europeans. To investigate the pathways leading to fragile X mutations in Africans, we expanded our previous sample of partially isolated communities of African ancestry (quilombos) investigating the populations of Abobral, Pedro Cubas, Galvão, and São Pedro in the Ribeira River Valley, state of São Paulo, South-Easthern Brazil (Fig. 1). Runaway slaves who reached the region between 1750 and 1850 founded these communities. A predominantly European-derived sample of 55 unrelated non-carrier brothers of fragile X males, collected in the city of São Paulo, was included in the study for comparison. All individuals were genotyped for DXS548 and FRAXAC1 alleles, as described by Mingroni-Netto et al. [2002]. Allelic frequencies at the two loci are shown in Table I. The frequencies of DXS548/FRAXAC1 haplotypes are in Table II. For AGG interruption analysis, a subset of 32 AfricanBrazilian males was selected, including individuals from Pontal, Cajual, and Rio das Rãs, previously studied by Mingroni-Netto et al. [2002]. Repeats on haplotypes known to be in linkage disequilibrium with the fragile X mutation either in Europeans (2-1 and 6-4) or in African-Americans (4-4 and 34) were preferentially sequenced, as well as those associated to uncommon haplotypes in European-derived populations. The 55 alleles from São Paulo city population were all sequenced, regardless of the linked haplotypes. In brief, the CGG repeat and surrounding DNA sequences were amplified from genomic DNA by Pfx polymerase (Invitrogen, Carlsbad, CA) with primers c and f [Fu et al., 1991]. In most samples, the forward and reverse strands were sequenced. Whenever sequencing of only one strand was obtained, the pattern was validated by re-sequencing. In the instances of rare or not previously reported patterns, sequencing was performed at least twice with each primer. After purification, approximately 60 ng of PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Forster City, CA), in an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer, Forster City, CA). CGG repeat interspersion patterns are presented in Figure 2. Haplotype 2-1 was not found among the four AfricanBrazilian populations from the state of São Paulo. In one sample, from Rio das Rãs, [Mingroni-Netto et al., 2002], this haplotype was linked to a 23 CGG repeat with one AGG interruption at the 50 end (Fig. 2a). In the predominantly Europeanderived sample from São Paulo city, the 2-1 haplotype was associated to four alleles with high repeat numbers ranging from 39–48 repeats. The 39 repeat had three interruptions: 9þ 9þ 9þ 9 (9 representing the number of CGG’s, and the signalþ one AGG interruption). The other alleles had two AGG interruptions: 9þ 9þ 22, 9þ 9þ 23, and 9þ 9þ 28 (Fig. 2b). Haplotype 6-4 was relatively frequent in the African-derived communities (8.3%–19.6%). It was less frequent in São Paulo city (1.8%, present study and 8.3%, Mingroni-Netto et al., 2002]. This is not a rare haplotype in populations hereto studied [Chiurazzi et al., 1996; Bonaventure et al., 1998; Jara Claudia B. Angeli and Leonardo P. Capelli equally contributed to this study.