Search

Background: Fragile X-associated tremor/ataxia syndrome (FXTAS), a novel disorder in male carriers of premutations of the fragile X mental retardation 1 gene (FMR1), was recently described. The clinical presentation of FXTAS most closely resembles multiple system atrophy (MSA) because both disorders manifest with cerebellar ataxia, intention tremor, autonomic dysfunction, and parkinsonism. It has been proposed that FXTAS might be a common neurodegenerative disorder., Objective: To determine whether FXTAS accounts for patients currently diagnosed as having MSA or a related clinical diagnosis., Design: Patients with MSA or related phenotypes were examined by experienced movement disorders neurologists, and DNA samples were obtained for genetic study., Setting: Salpêtrière Hospital., Patients: Seventy-seven patients clinically diagnosed as having MSA, 19 as having olivopontocerebellar atrophy, and 27 as having cerebellar ataxia., Main Outcome Measure: The number of FMR1 repeats was determined in all patients by polymerase chain reaction. Alleles above 40 CGG repeats were controlled by Southern blot analysis., Results: Two patients carried FMR1 premutations of 110 and 135 repeats: a man with a familial form of cerebellar ataxia and a woman diagnosed as having MSA-cerebellar type. In addition, 9 patients (7%) carried alleles in the intermediate size range, from 41 to 53 repeats., Conclusions: We confirm the recent initial description of FXTAS in women. Our data suggest that FXTAS is rare in MSA and indicate that FXTAS might be less prevalent than proposed.

Fragile X syndrome is associated with an unstable CGG-repeat in the FMR-1 gene. There are few reports of affected males transmitting the FMR-1 gene to offspring. We report on a family in which the propositus and his twin sister each had a full mutation with abnormal methylation. Their mother had an FMR-1 allele in the normal range and a large premutation, with normal methylation. The maternal grandmother had two normal FMR-1 alleles. The maternal grandfather had an unusual somatic FMR-1 pattern, with allele size ranging from premutation to full mutation. No allele was detectable by PCR analysis. Multiple Southern blot analyses identified a hybridization pattern that originated at a distinct premutation band and extended into the full mutation range. Methylation studies revealed a mosaic pattern with both unmethylated premutations and methylated full mutations. This individual declined formal evaluation but did not finish high school and has difficulty in reading and writing. The size of the premutation FMR-1 allele passed to his daughter is larger than his most prominent premutation allele. This is most likely due to gonadal mosaicism similar to that in his peripheral lymphocytes. Alternatively, this expansion event may have occurred during his daughter's early embryonic development and this large premutation allele is mitotically unstable. This pattern of FMR-1 alleles in a presumably mildly affected male is highly unusual. These findings are consistent with the absence of transmission of a full fragile X mutation through an expressing male. Studies of tissue specific FMR-1 allele expansion and FMR-1 protein expression on this individual should help to determine the correlation of the molecular findings with the phenotypic effects.

The pattern of inheritance in the fragile X (fra(X)) mutation follows a multistage intergenerational process in which the premutation evolves into the full mutation and the characteristic phenotype of the fra(X) syndrome after passing through oogenesis or a postzygotic event. Findings from our multicenter study confirm a strong direct relationship between fra(X) premutation size in the mother and probability of a full mutation in offspring with the mutation. Remarkably, the best-fitting equations are nonlinear asymptotic functions. The close approximation to both the logistic model and Gompertz suggests a process of accumulation of errors in DNA synthesis, as has been proposed previously. We also note that a larger-than-expected number of daughters of transmitting males have premutations that are smaller than their fathers', and that proportion is significantly higher than the proportion of daughters whose premutations are smaller than their mothers'. Intergenerational decreases in premutation size have been reported in other trinucleotide-repeat disorders and also appear to be parent-of-origin specific. Thus, while intergenerational expansion to the full mutation in fra(X) may manifest a postzygotic event, decreases in mutation size may occur during or prior to meiosis.

This study addresses mechanism of instability of the FMR-1 (CGG)n-repeat, and investigates features which may distinguish between normal stable and fragile X unstable repeats. To achieve this, we have sequenced 178 alleles to analyze patterns of AGG interruptions within the CGG repeat, and have typed the (CA)n-repeat at DXS548 for 204 chromosomes. Overall, our data is consistent with the idea that the length of uninterrupted CGG repeats determines instability. We predict that certain sequence configurations [no AGG, and (CGG)9-11AGG(CGG) > or = 20] present in the general population, are predisposed towards replication slippage. Association between these proposed predisposing repeats and DXS548 alleles may explain the previously reported frequencies of fragile X mutations and large-size normal repeats on specific haplotype backgrounds. We propose that predisposing alleles arise in the general population by as yet undefined mechanism(s) which introduce a relatively long stretch of pure CGG repeat at the 3'-end (relative to the direction of transcription) of the FMR-1 repeat region. The 3' pure repeat may then be susceptible to further expansion by replication slippage. Slippage on these predisposing chromosomes could accumulate over many generations until a threshold size is reached, at which point the repeat is susceptible to greater instability (i.e. premutation stage). Thus, results suggest that evolution of fragile X full mutations could involve 4 definable stages: 1) ancestral events leading to the formation of predisposing alleles which have large total repeat length (e.g. between 35 to 50) but no AGG or 1 AGG; 2) gradual slippage of these predisposing alleles to small premutations (S alleles); 3) conversion from S alleles to larger premutations (Z); 4) massive expansion from a Z allele to a full mutation (L).

Until recently, fragile X [fra(X)] syndrome was diagnosed by cytogenetic techniques and/or linkage analysis. Investigation of the mutation at the molecular level has shown that amplification of a polymorphic trinucleotide repeat (CGG) is diagnostic of this syndrome. Fu et al. [1991] observed that between 6-54 copies of the repeat were associated with alleles found in the general population, whereas 50-200 copies were associated with the premutation. In general, differences in copy number between the normal and premutated states are sufficiently large so that the probability of misclassification is, for all practical purposes, zero. However, there is a grey area in which members from both populations overlap. The purpose of our study was to determine the probability of misclassifying an individual from either the general or premutation population. DNA obtained from the general population and transmitting fra(X) females were analyzed from 3 centers in North America: Houston, Texas; Rochester, Minnesota; and Kingston, Ontario. The distribution of normal alleles from Houston was not significantly different from those obtained from Rochester. Therefore, these 2 samples were combined and the pooled distribution of normal alleles was compared with the pooled distribution of premutations. Results indicated that if 50 repeats were used as the cutoff criterion, sensitivity is 100%, specificity is 99.6%, and the probability that an individual has the fra(X) premutation given that the number of repeats is greater than 50 is 95%. Other cutoff criteria (45, 55, 60, 65) employed produced like findings, although 55 repeats appears to be a marginally superior criterion to 50. An independent sample from Kingston was used to verify the original assessments.(ABSTRACT TRUNCATED AT 250 WORDS)

Fragile X (fra(X)) males with a standardized IQ score of 70 or higher represent a high functioning (HF) or nonretarded fra(X) male group. This group, which does not include nonpenetrant males, has received little research attention to date. Of 221 fra(X) males who had been evaluated through The Children's Hospital in Denver since 1981 and had completed cognitive or developmental testing, 29 (13%) were high functioning by the above definition. We found that HF males on the whole had a lower cytogenetic score and were younger than retarded fra(X) males, but there was no difference between these two groups in the number of typical fra(X) physical manifestations present. FMR-1 DNA testing was performed on 134 fra(X) males and methylation status was determined for 51 of these. A greater percentage of HF males had a mosaic pattern or an incompletely methylated full mutation than did retarded males. A unique DNA pattern, an unmethylated fully expanded mutation, was discovered in 3 of the highest functioning fra(X) males. Protein studies performed on 2 of these males demonstrated the presence of FMR-1 protein, albeit at lower levels than normal. FMR-1 protein was not present in retarded fra(X) males. Significant FMR-1 protein expression may be responsible for higher cognitive functioning in the 2 males with unmethylated fully expanded mutations compared to retarded fra(X) males.

In this study, we have characterized a CGG repeat at the FMR-1 locus in more than 100 families (more than 500 individuals) presenting for fragile X testing and in 247 individuals from the general population. Both Southern blot and PCR-based assays were evaluated for their ability to detect premutations, full mutations, and variability in normal allele sizes. Among the Southern blot assays, the probes Ox1.9 or StB12.3 with a double restriction-enzyme digest were the most sensitive in detecting both small and large amplifications and, in addition, provided information on methylation of an adjacent CpG island. In the PCR-based assays, analysis of PCR products on denaturing DNA sequencing gels allowed the most accurate determination of CGG repeat number up to approximately 130 repeats. A combination of a Southern blot assay with a double digest and the PCR-sequencing-gel assay detected the spectrum of amplification-type mutations at the FMR-1 locus. In the patient population, a CGG repeat of 51 was the largest to be stably inherited, and a repeat of 57 was the smallest size of premutation to be unstably inherited. When premutations were transmitted by females, the size of repeat correlated with risk of expansion to a full mutation in the next generation. Full mutations (large repeats typically associated with an abnormal methylation pattern and mitotic instability) were associated with clinical and cytogenetic manifestations in males but not necessarily in females. In the control population, the CGG repeat ranged from 13 to 61, but 94% of alleles had fewer than 40 repeats. The most frequent allele (34%) was a repeat of 30. One female had an allele (61 repeats) within a range consistent with fragile X premutations, while two other individuals each had a repeat of 52. This suggests that the frequency of unstable alleles in the general population may be approximately 1%.

Introduction: Fragile X syndrome is the most commonly known inherited form of mental retardation. The intellectual abilities range from a normal IQ with learning disabilities to severe mental retardation. In males, there is a tendency for IQ decline in childhood. The purpose of this study was to correlate variations of the molecular cytosine guanine guanine (CGG) amplification in the fragile X mental retardation-1 (FMR-1) gene with the clinical findings, including IQ and physical features., Methods: Full-scale IQ and cytogenetic results in 116 individuals with the FMR-1 mutation were studied. The IQ testing was performed with age-appropriate standardized tests. Physical features were summarized in a physical index score for each patient. The FMR-1 results were determined with the OX1.9 probe and the following system was used: P1 indicates premutation; P2, large premutation to small full mutation; P3, full mutation; and P4, mosaic., Results/conclusions: The findings showed that those females with a small insert in the P1 range had a significantly higher IQ than other heterozygotes (P2, P3, and P4 categories). P4 males had a significantly higher IQ than P2 or P3 males. In cross-sectional age comparisons, the slope of the IQ decline was greater in P2 males than in P4 or P3 males.

In this study, 40 families segregating for fragile X [fra (X)] syndrome were examined for the presence of a mutation within the FMR-1 gene. Using the DNA probe Ox1.9, both carriers and affected individuals were found to contain an insertion/amplification-type of mutation with somatic instability. Variability in the size of the mutation, which ranged from less than 0.2 kb to approximately 13 kb, was observed both between individuals (even from the same family) and within individuals, who showed a smear rather than a discrete band(s) on Southern blot analysis. Transmission of the mutation by males resulted in little change of its size, while transmission by females usually resulted in an increase in size. Correlations were observed between the size of inserted/amplified DNA and the level of chromosome fragility and the presence or absence of mental impairment. Overall, a mutation was detected in 66 of 67 (99%) clinically affected males, in 12 of 13 (92%) transmitting males and in 95 of 112 (85%) carrier females. Equivocal results were obtained in 12 (11%) of the carrier females. No mutation was detected in 58 females and 33 males predicted to be normal by linkage, or in one female and 36 normal control males. These results strongly suggest that the mutation detected by Ox1.9 is closely associated with the cytogenetic and clinical expression of fra (X) syndrome. Additionally, the use of this probe along with other probe/enzyme combinations should provide a sensitive clinical assay for the detection of carriers of fra (X) syndrome.

Fragile X positive, mentally retarded males have been shown to have an insertion or amplification of DNA sequences at, or close to, the site of expression of the fragile site. We show here the application of the detection of such changes to the diagnosis of affected males and female carriers and the identification of normal transmitting males. One fragile X negative male with the clinical features of the Martin-Bell syndrome also possesses an inserted/amplified DNA sequence. The implications of these results for screening for the fragile X syndrome are discussed.

The fragile X syndrome locus, FRAXA, is located at Xq27. Until recently, few polymorphic loci had been genetically mapped close to FRAXA. This has been attributed to an increased frequency of recombination at Xq27, possibly associated with the fragile X mutation. In addition, the frequency of recombination around FRAXA has been reported to vary among fragile X families. These observations suggested that the genetic map at Xq27 in normal populations was different from that in fragile X populations and that the genetic map also varied within the fragile X population. Such variability would reduce the reliability of carrier risk estimates based on DNA studies in fragile X families. Five polymorphic loci have now been mapped to within 4 cM of FRAXA--DXS369, DXS297, DXS296, IDS, and DXS304. The frequency of recombination at Xq26-q28 was evaluated using data at these loci and at more distant loci from 112 families with the fragile X syndrome. Two-point and multipoint linkage analyses failed to detect any difference in the recombination fractions in fragile X versus normal families. Two-point and multipoint tests of linkage homogeneity failed to detect any evidence of linkage heterogeneity in the fragile X families. On the basis of this analysis, genetic maps derived from large samples of normal families and those derived from fragile X families are equally valid as the basis for calculating carrier risk estimates in a particular family.

The fragile X syndrome is the most common cause of familial mental retardation and is characterized by a fragile site at the end of the long arm of the X chromosome. The unusual genetics and cytogenetics of this X-linked condition make genetic counseling difficult. DNA studies were of limited value in genetic counseling, because the nearest polymorphic DNA loci had recombination fractions of 12% or more with the fragile X mutation, FRAXA. Five polymorphic loci have recently been described in this region of the X chromosome. The positions of these loci in relation to FRAXA were defined in a genetic linkage study of 112 affected families. The five loci--DXS369, DXS297, DXS296, IDS, and DXS304--had recombination fractions of 4% or less with FRAXA. The closest locus, DXS296, was distal to FRAXA and had a recombination fraction of 2%. The polymorphisms at these loci can be detected in DNA enzymatically digested with a limited number of restriction endonucleases. A strategy for DNA studies which is based on three restriction endonucleases and on five probes will detect one or more of these polymorphisms in 94% of women. This strategy greatly increases the utility of DNA studies in providing genetic advice to families with the fragile X syndrome.

We report on linkage data between DXS105, DXS98, the locus for the fragile X syndrome (FRAXA), and 3 other polymorphic loci that flank the FRAXA locus. An analysis was undertaken to determine the relative positions of DXS105 and DXS98 and to test the assignment of DXS105 to a location proximal and closely linked to FRAXA. In this study of fragile X fra(X) syndrome families, the DXS105 locus was calculated to be proximal to FRAXA with a maximum lod score of 10.36 at theta = 0.08. DXS105 was also shown to be closely linked to the gene for factor IX (F9)(Z = 11.84 at theta = 0.08) and to DXS98 (Z = 4.91 at theta = 0.04). The order of the loci proximal to FRAXA is most likely centromere-factor IX-DXS105-DXS98-FRAXA-telomere. The use of DXS105 and DXS98 in clinical investigations should significantly increase the accuracy of risk assessment in informative fragile X families.

We report the isolation and characterization of a novel DNA marker (1A1) in Xqter in the region of the fragile X. Genetic studies in families segregating for the fragile X syndrome suggest that 1A1 lies between the disease mutation and the distal locus, DXS52. Studies in normal and fragile X families show that 1A1 is tightly linked to DXS52 (Zmax = 17.20; theta max = 0.03) and F8 (Zmax = 7.01; theta max = 0.08). Multipoint mapping of families supports the order Xcen-DXS105-FRAXA-1A1-DXS52-(F8, DXS115)-Xqter. Pulsed-field gel electrophoresis (PFGE) studies demonstrate that 1A1 defines a new region of at least 2 Mb of DNA not physically linked to DXS52 or F8, thus extending the physical map of Xq27-qter to over 4 Mb. Complex partial digestion PFGE patterns, probably due to differing degrees of methylation, are observed with 1A1 in unrelated normal and fragile-X-positive individuals, whereas other distal markers give uniform digestion profiles. Physical data suggest that 1A1 lies in a region less CpG rich than other distal markers in Xq27-qter.

Linkage data, using the polymorphic markers 52A (DXS51), F9, 4D-8 (DXS98), and St14 (DXS52), are presented from 14 fragile X pedigrees and from 7 normal pedigrees derived from the collection of the Centre d'Etude du Polymorphisme Humaine. A multipoint linkage analysis indicates that the most probable order of these four loci in normal families is DXS51-F9-DXS98-DXS52. Recombination frequencies (theta) corresponding to maximum LOD scores (Z) were obtained by two-point linkage analysis for a number of linkage groups, including: DXS51-F9 (Z = 5.94, theta = 0.03), F9-DXS98 (Z = 0.51, theta = 0.26), F9-DXS52 (Z = 0.84, theta = 0.27), and DXS98-DXS52 (Z = 0.32, theta = 0.20). A multipoint linkage analysis of these loci, including the fragile X locus, was also performed for the fragile X population and the data support the relative order (DSX51, F9, DXS98)-FRAXA-DXS52. Recombination frequencies and maximum LOD scores, which again were derived from two-point linkage analyses, were obtained for the linkage groups DXS51-F9 (Z = 9.96, theta = 0) and F9-DXS52 (Z = 0.07, theta = 0.45) as well as for the groups DXS51-FRAXA (Z = 2.42, theta = 0.15), F9-FRAXA (Z = 1.30, theta = 0.18), DXS98-FRAXA (Z = 0.05, theta = 0.36), and DXS52-FRAXA (Z = 2.42, theta = 0.15). The linkage data was further tested for the presence of genetic heterogeneity both within and between the fragile X and normal families for the intervals DXS51-F9, F9-DXS52, F9-FRAXA, and DXS52-FRAXA using a modification of the A test. Except for the interval F9-FRAXA (P less than 0.10) there was no evidence of genetic heterogeneity for each of the various linkage groups examined. The heterogeneity detected for the interval F9-FRAXA, however, was most likely due to one family (Fx-28) that displayed very tight linkage between these two loci.

Recombinant DNA technology promises to play an increasingly important role in the future of medicine. Application of this technology to the study of human disease will help us to define and clarify the molecular pathology of many clinical disorders, provide new diagnostic tools and approaches, and, finally, will provide new therapeutic agents including gene-replacement therapy. We have begun to exploit these powerful new techniques to aid in the laboratory diagnosis of several genetic disorders for which reliable assays are currently not available, such as "Fragile X" syndrome.

A multilocus analysis of the fragile X (fra(X] syndrome was conducted with 147 families. Two proximal loci, DXS51 and F9, and two distal loci, DXS52 and DXS15, were studied. Overall, the best multipoint distances were found to be DXS51-F9, 6.9%, F9-fra(X), 22.4%; fra(X)-DXS52, 12.7%; DXS52-DXS15, 2.2%. These distances can be used for multipoint mapping of new probes, carrier testing and counseling of fra(X) families. Consistent with several previous studies, the families as a whole showed genetic heterogeneity for linkage between F9 and fra(X).

Eleven white families and 10 black families have been studied to detect racial differences in the linkage of DNA markers flanking the fragile X site (FRAXA). The differences in the recombination fractions for F9-FRAXA and DX13-FRAXA were not significant. The pair St14-FRAXA exhibited no difference between the two groups. Although the sample size was small, it would appear that these DNA markers can be used in black persons for prenatal diagnosis and genetic counseling. A larger group of families would be necessary to determine if 4D8 and cX55.7 will be equally useful since these appear to have lower heterozygote frequencies in the black population.