FRAXA syndrome

MARIA SYRROU 1, PHILIPPOS C. PATSALIS,2 IOANNIS GEORGIOU,1 YANNIS ALAMANOS,3 AND GERASSIMOS PAGOULATOS1

Abstract Microsatellites have been used for human evolution and origin studies by comparing their frequency, diversity, and allele size. In this study we report the analysis of three microsatellite loci, FMR] CGG and flanking DXS548 and FRAXAC2, in three separate groups of the Hellenic population: Athens, representing the general Hellenic population; Epirus (northwest Greece); and Cyprus. Significant variations in frequency and diversity were found in the three groups. Compared with Athens, Epirus had a tendency for longer alleles and a higher heterozygosity for DXS548. Cyprus had a frequency of CGG alleles similar to Athens but a low heterozygosity and a limited number of alleles at DXS548 and FRAXAC2. Allele differences of microsatellite loci not only are present in remote populations but also are evident between groups belonging to the same population. Microsatellite analysis could be a useful tool for identifying the origin of the founder chromosomes in intrapopulation studies and the time elapsed from the establishment of each population subgroup.

Microsatellites are polymorphic DNA sequences spread throughout the human genome (Weber and Wong 1993). Polymorphic microsatellites have been used for population studies of genetic variation because of the great variability of repeat number at most loci (Gilbert et al. 1990; Edwards et al. 1992). Certain trinucleotide repeats have recently attracted attention because of their association with disease genes. The CGG trinucleotide expansion in the FMRI gene is the cause of fragile X syndrome. The number of the CGG repeat sequence in normal individuals is highly polymorphic, with a range of 5-50 repeats (Fu et al. 1991; Kremer et al. 1991). The most common alleles reside between 29 and 32 repeats, and there are secondary peaks at 19 to 23 repeats (Fu et al. 1991; Snow et al. 1993; Brown et al. 1993; Chiurazzi et al. 1996; Murray et al. 1996).

A number of population genetic studies have suggested similar distribution of CGG repeats of the FMRI gene in various populations (Fu et al. 1991; Snow et al. 1993; Brown et al. 1993; Chiurazzi et al. 1996; Murray et al. 1996). Although distributions invoke a similar trend in the evolution of CGG repeats in human populations studied so far, there are differences in the relative frequency of the primary and the secondary peaks between the studies (Fu et al. 1991; Brown et al. 1993). Asian, European, and African populations appear to have some distinguishable differences in the number of repeats (Chiurazzi et al. 1996; Zhong et al. 1994).

The flanking microsatellite markers DXS548 and FRAXAC2 have been studied to provide additional evidence of the distribution of certain (CGG)n alleles in various populations (Richards et al. 1991; Riggins et al. 1992; Verkerk et al. 1991; Oudet et al. 1993; Macpherson et al. 1994; Malmgren et al. 1994; Syrrou et al. 1996). These markers are within FMRI, are located 150 kb and 10 kb, respectively, from the CGG repeat, and show no recombination with FRAXA or with one another (Richards et al. 1992).

Thus we compare the distribution and the relative frequencies of CGG alleles, DXS548 and FRAXAC2 alleles, and their haplotypes for three different groups in the Hellenic population [the general Hellenic population (Athens), Epirus (northwest Greece), and Cyprus] to study within-population variation.

Materials and Methods

Samples for CGG allele analysis were obtained from families referred for genetic services because of single gene or chromosomal defects. The individuals were selected to be normal in the CGG repeat number to avoid founder effects or fragile X syndrome patients. Chromosomes from 199 normal individuals (75 male and 124 female) of Hellenic origin coming from different parts of Greece and Cyprus were studied (Figure 1). This population was divided into three groups: (1) general population [126 chromosomes from individuals originating from various regions of Greece and having genetic services in Athens (designated as Athens)], (2) Epirus [105 chromosomes from Epirus (northwest Greece)], and (3) Cyprus (92 chromosomes from Cyprus). CGG data from 323 chromosomes are included. Here, we compare the (CGG)n distribution of normal individuals in two different distal populations of Hellenic origin with corresponding individuals from the general Hellenic population.

One hundred seven chromosomes obtained from 75 male and 16 female individuals from the total sample were analyzed for both microsatellite markers (DXS548 and FRAXAC2) and haplotypes resulting from the combination of the two microsatellite markers (DXS548-FRAXAC2): 36 from the general population, 33 from Epirus, and 38 from Cyprus (Table 1). An additional number of 32 parents were studied for the appropriate determination of haplotypes in females.

CGG repeats were amplified using a nonradioactive polymerase chain reaction (PCR), separated on denaturing gels, transferred to nylon filters, and detected according to the method of Brown et al. (1993). PCR amplification of CA polymorphisms was performed with oligonucleotide primers described by Verkerk et al. (1991) for the DXS548 locus and by Richards et al. (1992) for the FRAXAC2 locus after end-labeling of one primer with 32p. Products were diluted in formamide and separated on 6% polyacrylamide gel with the appropriate molecular weight markers.

Statistical analysis was done using the sPss program. Significance of intrapopulation variation of alleles was estimated using the chi-square test. Fisher's exact test and Yates correction were implemented when appropriate. Alleles and haplotypes with low or zero frequencies were grouped together to apply the statistical test.

Results

Among the three Hellenic populations of Epirus, Cyprus, and Athens statistically significant differences were observed in the overall distribution of (CGG)n alleles (p

Statistically significant differences between the three population groups were observed in the distribution of DXS548 alleles (p 40) was observed.

All DXS548 alleles of the Hellenic population were represented in Athens, whereas in Epirus alleles *192, *200, and *206 and in Cyprus alleles *196, *200, and *206 were absent. Twelve haplotypes were found in Athens, whereas 7 and 9 haplotypes were found in Epirus and Cyprus, respectively. Haplotypes 192,151, 194,149, and 204,151 were found only in Cyprus. There is also a tendency toward longer alleles in the Epirus population compared with the Cyprus population in the three loci (Table 1, Figures 2 and 3).

Table 2 presents the expected heterozygosities at every locus, demonstrating that the three population groups have comparable CGG and FRAXAC2 heterozygosities. Distribution of alleles and haplotypes was similar for males and females. Discussion

Recent studies of allele variation and distribution of microsatellites in humans and nonhuman primates (Cavalli-Sforza 1991; Cavalli-Sforza and Piazza 1993; Rubinsztein et al. 1995) suggest that in humans longer alleles are the result of evolution. In particular, surveys for (CGG)n repeats in the FMRI gene and (CAG)n repeats in the Huntington disease gene and their flanking (CA)n haplotypes have shown that trinucleotide alleles expanded from shorter ancestral alleles, exhibit asymmetric distributions, and have differences in frequency between remote populations (Zhong et al. 1994; Chiurazzi et al. 1996; Eichler and Nelson 1996).

Microsatellite variability has been useful for interpopulation studies (Gilbert et al. 1990; Bowcock et al. 1994). Surveys based on microsatellite loci analysis in different human populations have established evolutionary relationships between these populations (Cavalli-Sforza 1991; Kidd et al. 1991; Cavalli-Sforza and Piazza 1993). Microsatellite variability and gene diversity are attributed to the time elapsed since founding and to the population size in the presence of constant mutation rates and absence of selection pressure (Paabo 1995). Heterozygosity has also been suggested to be an index of the time elapsed from the founding of a given population (Kidd et al. 1991; Bowcock et al. 1994).

We have used microsatellites (CGG, DXS548, FRAXAC2) to identify variations in gene distributions and frequencies among three different groups in the Hellenic population. FRAXAC2 and DXS548 show no recombination with FMRI or with one another and are consequently reliable markers for variation at the FMRI locus. Our data demonstrate that there is a tendency toward longer alleles in DXS548, FRAXAC2, and FMRI (CGG)n loci in Epirus and a relative absence of long alleles in Cyprus (Table 1). There is also a significantly higher heterozygosity at DXS548 in Epirus.

Although the population size is small and the three population groups belong to the same general population, differences are evident. We have observed higher heterozygosity in at least one of the microsatellite loci (DXS548). This observation might be consistent with other evidence that greater genetic diversity is correlated with more ancient establishment or origin (Bowcock et al. 1994; Deka et al. 1995). The Cyprus population exhibits genetic homogeneity. Additional evidence for this homogeneity is that haplotype 194,153 is the most prevalent and is related to the highest number of CGG repeats in this population. This observation is supported by the limited number of fi-thalassemia alleles in Cyprus compared with the spectrum of betathalassemia alleles in Greece and the rarity of cystic fibrosis mutations (Baysal et al. 1992; Boteva et al. 1994).

The absence of a secondary peak in the (CGG)n distribution in Epirus and Cyprus may be the result of the small population size, which reduces variability (Paabo 1995). The existence of long CGG alleles (>20 repeats) in Epirus and Cyprus suggests that longer alleles have a higher representation rate in the studied small population subgroups compared with the general population.

Microsatellite analysis could be a useful tool for identifying the origin of the founder chromosomes not only among unrelated populations but also in intrapopulation studies. Acknowledgments This study was supported in part by the General Secretariat of Research and Technology, Ministry of Development, Greece. Received 29 October 1996; revision received 3 June 1997.

1 Laboratory of General Biology, Medical School, University of Ioannina, 45500 Ioannina, Greece. 2Department of Cytogenetics, Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus. 3 Department of Hygiene, Medical School, University of Ioannina, Ioannina, Greece.

KEY WORDS: MICROSATELLITES, INTRAPOPULATION VARIATION, FMRI

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