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The human fragile-X syndrome is associated with expansions of a (CGG)n triplet repeat within the FMR1 gene. Whilst normal FMR1 arrays consist of variable numbers of (CGG)7-13 blocks punctuated with single AGG triplets, unstable arrays contain longer blocks of uninterrupted (CGG)n. The degree of instability, and subsequent risk of expansion to the fragile-X mutation, is dependent upon the length of this uninterrupted repeat. Detailed analyses of normal FMR1 array structures suggest that longer uninterrupted blocks of repeat could arise either through a process of gradual slippage or a more dramatic loss of an intervening AGG triplet. Up to 15% of Japanese and Chinese individuals have FMR1 triplet arrays centred on 36 repeats in length, a modal group not found in Caucasians. As longer FMR1 arrays have been associated with high-risk fragile-X haplotypes in some populations, we investigated the nature of these larger arrays. Sequence analysis revealed that the unusual length is due to the presence of a novel (CGG)6 block within the array. Several haplotypically related arrays contain blocks of (CGG)16 or (CGG)15, consistent with the fusion of adjacent (CGG)9 and (CGG)6 blocks after loss of the intervening AGG triplet. This is compatible with inferences from the Caucasian population that AGG loss is a mechanism by which long blocks of identical repeats are generated.

The fragile X syndrome is predominantly caused by a large expansion of a CGG trinucleotide repeat in the promoter region of the FMR1 gene, which is associated with methylation and downregulation of transcription. The molecular diagnosis of this disorder is based on repeat size and methylation analysis of the FMR1 gene usually by Southern blot analysis. We describe a PCR-based method for the analysis of methylation of the FMR1 gene, which involves bisulfite treatment of DNA prior to amplification. Fifty-two normal and 48 affected, premutation, or mosaic males were analyzed in a blinded study by this method. A prospective study of 30 males suspected of fragile X was also performed. Amplification specific for the methylated FMR1 sequence was readily observed in all individuals with a full mutation, whereas all normal and premutation individuals showed only amplification-specific for the unmethylated sequence, thus, allowing affected and unaffected males to be distinguished. A full mutation in the presence of mosaicism was also detectable by this method. Methylation-specific PCR appears to be a rapid and reliable tool for the diagnosis of fragile X males.

Longitudinal declines in IQ among fragile X [fra(X)] males have been reported previously by several investigators. Remarkably little is known about longitudinal changes in IQ scores among fra(X) females. Previously, one cross-sectional study showed a significant negative correlation between age and IQ scores. However, a recent investigation of girls with fra(X) syndrome noted longitudinal increases in IQ scores in 8 of 11 individuals. Therefore, the purpose of this preliminary multicenter study was to determine: (1) the characteristics of longitudinal changes in IQ among fra(X) females; and (2) whether these changes were comparable to those which have been observed among fra(X) males. IQ test and retest scores for 11 fra(X) females were obtained from 3 centers: Greenwood, South Carolina; Ibaraki, Japan; and Leuven, Belgium. To ensure high reliability, only test-retest scores from the Wechsler and Stanford-Binet tests were used. Age of subjects at initial testing ranged from 5 to 35 years. Mean intertest interval was 4.5 years. In contrast to a report of longitudinal increases, we found 9/11 (82%) subjects demonstrated decreases in IQ scores. Mean decline was 9 points. Females over 18 years of age showed little or no change in IQ scores. Decreases in scores appeared to be related to initial IQ score. Females in the earlier longitudinal report were higher functioning than those in our study, which may account for the observed difference in direction of change; or, change in IQ score may be related to size of the fra(X) mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

The prevalence of the fragile X syndrome has been estimated by the results of population studies in which the disease was mostly diagnosed by cytogenetic examinations. To examine the reliability of the cytogenetic analysis for the estimation of the prevalence of the fragile X syndrome, the CGG repeat in the FMR-1 gene was assayed by Southern blot hybridization and polymerase chain reaction (PCR) in an institutionalized group of mentally retarded individuals consisting of 305 males and 129 females. The data thus obtained were compared with the cytogenetic data. The DNA analysis detected 7 full mutations among the alleles of the 296 unrelated males and 2 full mutations among the alleles of the 129 unrelated females. These findings were consistent with the cytogenetic data. No premutation was found in 554 X chromosomes in the unrelated mentally retarded patients nor 826 X chromosomes in unrelated control individuals. The distribution of the CGG repeat number in the normal range was not significantly different between the mentally retarded individuals and the control individuals. These data suggest that the estimates of the prevalence of the fragile X syndrome based on cytogenetic data in the population studies are almost reliable. Based on the finding that no premutations were found in this study, a small difference in the prevalence of the fragile X syndrome between Caucasians and Japanese is suggested.

The fragile X mutation is the result of amplification in the repeat number of p(CGG)n in FMR-1; alleles with more than 52 repeats have been shown to be so unstable as to mutate in the repeat number in almost every transmission. To improve our understanding of mutations in normal alleles of FMR-1, the following studies were carried out in the Japanese population: a study on length variation in the repeat to determine the allele distribution of the repeat length in a non-retarded population, family studies to observe new mutations in normal allele, and haplotype analyses with microsatellite markers flanking the repeat to confirm estimated mutation rates and founder chromosomes in the fragile X syndrome. Analysis of the p(CGG)n in 370 unrelated males detected 24 distinct alleles with repeats of 18-44. A comparison with previously reported data suggests the presence of racial/ethnic differences in the allele distribution. No premutation allele was found in 824 unrelated X chromosomes examined by the polymerase chain reaction and Southern blot analysis. Family studies detected one new mutation in a total of 303 meioses. However, the mutation rate was not in accordance with the expected or observed heterozygosities in the population or with linkage disequilibrium observed between the repeat numbers and the haplotypes of the markers flanking the CGG. The haplotype in the chromosome in which the new mutation was found was the same as that frequently found in the Japanese fragile X chromosomes, and the variance in the CGG repeat number was wider in chromosomes with the haplotypes frequently found in the fragile X chromosome than in those with the other haplotypes. These observations suggest that a subgroup is present in normal alleles and that this subgroup is more liable to mutate than others.

Longitudinal changes in IQ among mentally retarded (MR) fragile X [fra(x)] males have been reported previously. While age is associated with decline in IQ, not all males are so affected. This suggests that there may be more than one subtype of affected fra(X) male. Therefore, we examined the distribution of standardized difference scores (Zdiff) in IQ to determine if subjects were from an admixture of at least 2 populations. Cluster analysis of Zdiff scores was used to partition subjects into 2 groups. Goodness-of-fit tests indicated that scores were more likely to come from an admixture. Discriminant functions (DF) were calculated to determine predictive validity of Zdiff scores. To eliminate the effect of skewing, a power transform was applied to Zdiff scores and DFs recomputed. Zdiff and transformed scores provided similar results. The mean and variance for one group showed no differences in test-retest scores as would be expected from examining any population while the mean for the second group indicated significant decline in IQ nearly 4 standard errors below the first test score. These results suggest that there may be clinical evidence for 2 types of fra(X) mutation: One which causes MR but is static, and a second mutation which causes MR but is dynamic and contributes to an apparent longitudinal decline in cognitive function.

Longitudinal decline in IQ among fragile X males was reported recently. However, there are problems in retesting IQ that may affect scores. Two such factors are intertest time interval and score obtained on the first test. To determine the generality of IQ score changes, we examined 101 fragile X males from 6 centers. To ensure high test-retest reliability, only results from Stanford-Binet and Wechsler tests were used. Thus there were retest scores from 60 subjects. Test-retest reliability between first and last scores was very good (r = 0.85) and comparable to those seen in nonfragile X mentally retarded individuals. Also computed were z-scores of differences in IQ scores. The z-score differences were distributed about a mean at 1 SD below the expected zero value. Eighteen subjects showed statistically significant decreases in IQ, 6 showed statistically significant increases, while 5 showed the same scores. Z-score differences were not correlated with type of residence or elapsed intertest interval, but were negatively correlated with first score obtained, indicating a regression-to-the-mean effect. Using a multiple regression analysis, we found first score obtained, age tested, and age retested significant predictors of score differences, accounting for 19% of the total variance. These results suggest that factors previously identified as affecting retest scores have a smaller effect than originally thought. It is suspected that decline in IQ is associated with dynamic neurological processes and needs to be investigated further.

The replication status of the fragile X chromosomes was studied in short-term cultures of lymphocytes from six female heterozygous carriers. The fragile X was induced by adding 0.1 microM fluorodeoxyuridine during the last 24 h of culturing. The replication status of the X chromosomes was studied using a bromodeoxyuridine (BrdUrd) antibody method. BrdUrd was added (1) at a final concentration of 0.2 micrograms/ml during the early S phase of chromosome replication (16-10 h before harvest), (2) at 0.2 microgram/ml during the late S phase (the last 6 h of culturing), (3) at 20 micrograms/ml during the early S phase, and (4) at 20 micrograms/ml during the late S phase. BrdUrd that was incorporated into replicating chromosomes was detected by using a nuclease and BrdUrd monoclonal antibody. The frequency of the fragile X was reduced by BrdUrd treatment. The degree of reduction was more severe in the 20 micrograms/ml than in the 0.2 microgram/ml series and was more severe with late S than with early S treatment. Of the early- and late-replicating fragile X chromosomes, those which were actively replicating during a BrdUrd treatment were more reduced than the others. Thus, the average rate of early and late S treatment with 0.2 microgram BrdUrd/ml was assumed to be the closest reflection of the situation in vivo. There was no correlation between the average rate of the early replicating, active fragile X and the intelligence of the heterozygous carriers studied.

Among 243 institutionalized mentally retarded males in Japan, 13 patients (5.3%) with the fra(X)(q27) from nine families were detected. These 13 patients accounted for 8.6% of 152 male inmates with unknown causes of mental retardation in the population. One out of nine pedigrees had an apparently unaffected male transmitter of this disorder. Our data agree with the frequencies of the fra(X) syndrome in various retarded populations, most of which were Caucasians, suggesting that the prevalence of the fra(X) syndrome in Japanese is not significantly different from those in Caucasians.

Clinical and cytogenetic aspects of a female infant with trisomy 21 and the fragile X [fra(X)] chromosome are reported. Most of the facial characteristics of the patient are those observed in Down syndrome, but some features such as long face with prominent forehead and lower jaw, and large ears are related to the fra(X) syndrome. The origin of an additional chromosome 21 may be ascribed to maternal first meiotic nondisjunction in our case. It has been suspected that female carriers of the fra(X) chromosome may be predisposed to meiotic nondisjunctional events. However, there is probably no relationship between the two chromosomal abnormalities in our case because of the maternal age at the delivery.

Auditory brain-stem responses (ABRs) were recorded from a group of 12 mentally retarded males with the fragile X (fra[X]). The responses were analyzed in terms of ABR thresholds, absolute latencies, and interpeak latencies. One patient had increased ABR thresholds, indicating hearing impairment. Five fra(X) subjects had prolonged I-V interpeak latencies. Comparisons between the fra(X) group (excluding one possible hard-of-hearing subject) and a control group of age-matched males with normal intelligence showed that the fra(X) group's interpeak latencies were significantly prolonged for the III-V and I-V but not for the I-III. This pattern of prolongation of interpeak latencies suggests that central, as opposed to peripheral, nervous-system dysfunction predominates in many patients having this syndrome. In addition, frequently observed prolongation of the transmission time may indicate that brain-stem white-matter functioning is also apt to be involved in this syndrome.

The fragile X [fra(X)] syndrome was screened on 190 Japanese institutionalized females with moderate to severe mental retardation. Two inmates with severe mental retardation (IQ 20) had the fra(X) chromosome in 26% and 15% of the cells examined, indicating that the prevalence of the fra(X) syndrome was about 1% in all female inmates and was about 3.27% in severely mentally retarded females without known causes. However, no female with fra(X) syndrome was found in 35 moderately retarded females. Both had brothers with the fra(X) syndrome and the prevalence was 10% in females with a family history of mental retardation. In addition, the replication study of the fra(X) chromosome in the patients supported the proposal that an excess of the early replicated fra(X) chromosome is related to the mental capacity in heterozygous females. Therefore, the fra(X) syndrome should not be ignored even in severely mentally retarded females with a family history, though the heterozygotes are commonly normal to subnormal in their mental development. In addition, the replication study of the fra(X) chromosome may help to estimate mental development in the carrier children.