Factors involved in the stability of trinucleotide repeats during transmission were studied in 139 families in which a full mutation, premutation or intermediate allele at either FRAXA or FRAXE was segregating. The transmission of alleles at FRAXA, FRAXE and four microsatellite loci were recorded for all individuals. Instability within the minimal and common ranges (0-40 repeats for FRAXA, 0-30 repeats for FRAXE) was extremely rare; only one example was observed, an increased in size at FRAXA from 29 to 39 repeats. Four FRAXA and three FRAXE alleles in the intermediate range (41-60) repeats for FRAXA, 31-60 for FRAXE) were unstably transmitted. Instability was more frequent for FRAXA intermediate alleles that had a tract of pure CGG greater than 37 although instability only occurred in two of 13 such transmissions: the changes observed were limited to only one or two repeats. Premutation FRAXA alleles over 100 repeats expanded to a full mutation during female transmission in 100% of cases, in agreement with other published series. There was no clear correlation between haplotype and probability of expansion of FRAXA premutations. Instability at FRAXA or FRAXE was more often observed in conjunction with a second instability at an independent locus suggesting genomic instability as a possible mechanism by which at least some FRAXA and FRAXE mutations arise.

There have been several claims of segregation distortion (meiotic drive) for loci associated with diseases caused by trinucleotide repeats, leading us to test for this phenomenon in a large study of the X-linked loci FRAXA and FRAXE. We found no evidence of meiotic drive in females and no convincing evidence in males, where the limitation of risk to daughters creates a testing bias for alleles of interest. Alleles for pre-and full mutation, intermediate alleles, and common alleles were analyzed separately, with the same negative results that are extended in the discussion to claims of meiotic drive for other diseases. Meiotic drive, or segregation distortion, is a rare and incompletely understood phenomenon resulting in a breach of Mendel's second law and inheritance of an excess, and often a very large excess, of one class of gamete (1). Several reports in man, involving a number of different autosomal loci, have suggested preferential inheritance of one class of gamete, generally that carrying the mutant allele, from parents of one sex but not the other (e.g., refs. 2-4). These claims of segregation distortion usually are based on small numbers of observations and, where the observations are reported in sufficient detail, often can be seen to be the result of failure to correct for ascertainment bias.Recently, there have been a number of papers suggesting the occurrence of segregation distortion in the transmission of loci associated with diseases caused by trinucleotide repeat expansions. Because this class of mutation is novel and does not obey the rules of Mendelian inheritance in a number of respects, it might also defy Mendel's law on segregation. The most persistent reports of meiotic drive in a trinucleotide repeat disease have been associated with myotonic dystrophy. Carey et al. (5) reported that among normal individuals with one allele at the upper end of the normal range (Ն19 repeats), the large allele was selectively transmitted during male, but not female, meiosis. This suggested to the authors that transmission favoring large and perhaps more mutable alleles was a mechanism for maintaining myotonic dystrophy in the population in spite of its reduced reproductive fitness. Gennarelli et al. (6) reported a similar excess of individuals with myotonic dystrophy expansions in the affected range among children of affected parents, the excess being particularly marked in children of affected males. However, both these observations were criticized on statistical grounds by Hurst et al. (9) studied individual spermatozoa from three men, each of whom had one large myotonic dystrophy allele with a repeat number greater than 19. In no instance did they find a transmission ratio significantly different from 0.5. Ikeuchi et al. (10) claimed segregation distortion in both dentatorubral-pallidoluysian atrophy (DRPLA) and Machado-Joseph disease with preferential transmission of the large alleles following male, but not female, meiosis in both diseases. In these analyses, alleles in the pathological siz...

FRAXE full mutations are rare and appear to be associated with mild mental retardation. As part of a screening survey of boys with learning difficulties to determine the frequency of full and premutations, we have collected data on the frequency of instability at FRAXE for about 4000 transmissions and the haplotype for over 7000 chromosomes. The distribution of FRAXE repeats was similar to other English populations but differed from two North American Caucasian series. Observed instability at FRAXE was rare but increased with increasing repeat number, and there were no expansions into the full mutation range, except in pedigrees ascertained through a full mutation. Haplotype analysis suggested division into five groups with each group having a characteristic distribution of FRAXE repeats. Fourteen of the 15 full mutations occurred on a single haplotype and this haplotype also had a significant excess of intermediate-sized alleles, suggesting that full mutations originate from large normal alleles. However, a related haplotype also had a significant excess of intermediates but we observed no full mutations on this haplotype, suggesting either loss or gain of stability determinants on it. We suggest that whilst triplet repeat size is a significant predisposing factor for expansion at FRAXE other genetic determinants are also likely to be important.

Studies on the population genetics of fragile X syndrome have relied on haplotyping microsatellite markers closely flanking the FMR1 gene on normal and fragile X chromosomes. Strong linkage disequilibrium between the fragile X mutation and a few haplotypes have been reported in several populations, suggesting the occurrence of a limited number of initial mutational events [Chiurazzi et al., 1996a]. The three microsatellite repeats most frequently employed in haplotyping are DXS548 [Riggins et al., 1992], FRAXAC1, and FRAXAC2 [Richards et al., 1991]. Different systems have been used by various groups for identifying the numerous alleles at each of these marker loci, thus impeding reliable comparisons of the results across populations and conclusions on the identity of fragile X "founder chromosomes." In fact, several authors attempted to align the allele distributions of the flanking microsatellites by assuming that the mode(s) in populations of European origin were the same [Chiurazzi et al., 1996a;Macpherson et al., 1994;Rousseau et al., 1995]. However, this "alignment" procedure of different distributions cannot be considered satisfactory and an absolute reference must be introduced at some point. Unfortunately, sizing of polymerase chain reaction (PCR) products by running them next to markers or even sequences (e.g., M13) derived from other loci is not a reliable method at the single base-pair resolution level, because the different GC content and secondary structures make fragments of the same length migrate with slight differences. Thus, only one (or more) allele(s) of the same microsatellite which have been actually sequenced would constitute such an absolute reference. However, sequence information for all three loci obtained from the same samples is not yet available, and considering that sequencing of repeated sequences can be troublesome and that different centers may obtain different results, we first decided to genotype a few lymphoblastoid cell lines for these markers and to start distributing their DNA to all interested laboratories. Therefore, we have established a panel of DNA samples derived from several cell lines maintained in our laboratories and that we are intending to sequence. It is important to realize that, even in the absence of sequence information, the sharing of reference DNAs will allow comparison of the results obtained in the various FMR1 haplotype studies. All DNA samples (seven males and one female for a total of eight) were genotyped for DXS548, FRAXAC1, and FRAXAC2 independently by each participating group in Italy, the United Kingdom, and Canada, respectively. This letter to the editor is intended to call the attention of fellow researchers who wish to replicate our sequencing effort and to inform them that this panel is available freely to all researchers and can be requested at the Internet address http://www.rmga.qc. ca/panel/. Upon request via the WWW site, the closest reference laboratory will provide two or three representative DNA samples from the panel. Ideally, all ...