FMR1 fully expanded mutation with minimal methylation in a high functioning fragile X male.

Cytogenetic and molecular genetic analysis of a peripheral blood sample from a 31 year old, non-mentally retarded male with a family history of fragile X syndrome showed unexpected results. Nine percent of cells evaluated cytogenetically expressed a fragile X chromosome and molecular examination of the FMR1 gene showed a highly unusual pattern defined as a minimally methylated fully expanded mutation. This case illustrates the need to recognise exceptional variations of fragile X syndrome mutations.     

Effect of the fragile X status categories and the fragile X mental retardation protein levels on executive functioning in males and females with fragile X

The effects of a fragile X disorder on executive function impairment were assessed in 144 extended families, which included individuals with fragile X premutation and full mutation and their relatives without fragile X. A modification of the maximum-likelihood estimators for pedigree data, as well as ordinal logistic regression, were used in data analysis. The most outstanding deficit, occurring especially in males, involved impaired capacity to use an intention to regulate purposeful behavior. This deficit occurred independently of general cognitive impairment but was related to depletion of fragile X mental retardation 1 gene protein product. The other executive function deficits were accounted for by the general cognitive impairment. Possible mechanisms of the effect of fragile X premutation on impairments of executive functioning are considered.     

Cognitive-behavioral profiles of females with the fragile X mutation

The fragile X (FRAXA) mutation is typically manifested as either a full mutation (FM) or premutation (PM), and is often associated with some form of learning impairment. The study by Lachiewicz et al. in this issue suggests that females with the FM or PM exhibit a specific profile of strengths in verbal abilities and significant weaknesses in quantitative skills. We examined 17 females with either the FM or PM using a standard cognitive-behavioral battery consisting of the Stanford-Binet (4th Edition; SBFE) and the Vineland Adaptive Behavior Scale (VABS). Although we found the expected differences in composite IQ scores and adaptive behavior composite scores (DQ) between FM and PM females, we found no significant differences between verbal and quantitative reasoning in either group.     

Investigation of the segregation of the fragile X mutation in daughters of obligate carrier women

Two reports have suggested that over 50% of the offspring of obligate carrier women receive the mutation for the fra(X) or the Martin-Bell syndrome [Webb et al, 1986; Fryns, 1984]. Such a segregation distortion is difficult to assess for the fra(X) syndrome because of incomplete penetrance, variable expression and probable ascertainment biases. We have attempted to evaluate this possible segregation distortion in daughters of obligate carriers in a large sample of sibships ascertained in a survey of New South Wales, Australia. We used two definitions of expression: 1) presence of fra(X) positive cells if daughters were tested cytogenetically, and 2) mental impairment if daughters were not tested cytogenetically. The segregation frequency was estimated in different types of sibships of obligate carriers based on the way they were ascertained. This was done in order to have an internal check on possible ascertainment biases. Among the 189 cytogenetically tested daughters, 81 were fra(X) positive. Among the 97 untested daughters, 24 were mentally impaired in some way. Therefore, the segregation frequency as defined by fra(X) expression and/or mental impairment was 37%. Thus, no evidence was detected for segregation distortion. These data were significantly different than those collected by Webb et al [1986] and scored by the same method as the present data set.     

Origins of the fragile X syndrome mutation.

The fragile X syndrome is a common cause of mental impairment. In view of the low reproductive fitness of affected males, the high incidence of the syndrome has been suggested to be the result of a high rate of new mutations occurring exclusively in the male germline. Extensive family studies, however, have failed to identify any cases of a new mutation. Alternatively, it has been suggested that a selective advantage of unaffected heterozygotes may, in part, explain the high incidence of the syndrome. Molecular investigations have shown that the syndrome is caused by the amplification of a CGG trinucleotide repeat in the FMR-1 gene which leads to the loss of gene expression. Further to this, genetic studies have suggested that there is evidence of linkage disequilibrium between the fragile X disease locus and flanking polymorphic markers. More recently, this analysis has been extended and has led to the observation that a large number of fragile X chromosomes appear to be lineage descendants of founder mutation events. Here, we present a study of the FRAXAC1 polymorphic marker in our patient cohort. We find that its allele distribution is strikingly different on fragile X chromosomes, confirming the earlier observations and giving further support to the suggestions of a fragile X founder effect.     

Sequence analysis of the fragile X trinucleotide repeat: implications for the origin of the fragile X mutation

This study addresses mechanism of instability of the FMR-1 (CGG)_n-repeat,and investigates features which may distinguish between normalstable and fragile X unstable repeats. To achieve this, we havesequenced 178 alleles to analyze patterns of AGG interruptionswithin the CGG repeat, and have typed the (CA)_n-repeat at DXS548for 204 chromosomes. Overall, our data is consistent with theidea that the length of uninterrupted CGG repeats determinesInstability. We predict that certain sequence configurations[no AGG, and (CGG)_9–11AGG(CGG)     

Methylation and mutation patterns in the fragile X syndrome.

Chromosomes carrying the mutation causing the fragile X [fra(X)] syndrome have been shown to have an unstable DNA sequence close to or within the fragile site. The length variation is located within a DNA fragment containing a CGG trinucleotide repeat which is unstable in both mitosis and meiosis. We have used the probe StB12.3 from the region to analyze the mutations and the methylation patterns in 21 families segregating for the fra(X) syndrome. Among 40 fra(X) males all showed an abnormal pattern. The normal 2.8 kb band was absent in 36 individuals and replaced by a heterogeneous smear of larger size. The remaining four were shown to be "mosaics" with the presence of both mutated, unmethylated and mutated, methylated fragments. We found four normal transmitting males, one which was a great-grandson of another normal transmitting male indicating that the pre-mutation can remain stable through two meioses in the female. In nine fra(X) positive females the abnormal pattern consisted of a smear, usually seen in affected males, in addition to the normal bands. Five of these females were mentally normal. Of clinical importance is the prediction of mental impairment in females. We suggest that this is not made by the detection of the full mutation alone, but rather by the degree of methylation of the normal X chromosome. Our results suggest that difference of clinical expression in monozygotic twins may be correlated with difference in methylation pattern. Six out of 33 fra(X) negative females at risk were diagnosed as carriers. Our observations indicate that molecular heterogeneity is responsible for variable expression of the fra(X) syndrome in both males and females.     

Adaptive behavior in the fragile X syndrome: Profile and development

In this study we present data on the adaptive behavior profile and on the development of adaptive functioning in 39 fragile X [fra(X)] males, age 4–26 years. Social adaptability is relatively well developed as compared to cognitive level and especially self-help skills continue to grow with age despite a stagnation in intellectual growth. © 1993 Wiley-Liss, Inc.     

Expand Long PCR for fragile X mutation detection

Fragile X mutation detection by DNA analysis enables accurate diagnosis of the fragile X syndrome. The mutation involves the expansion of CGG repeats in the FMR1 gene and has been primarily detected by the Southern blotting method. In this study we present a novel, efficient and reliable PCR protocol that is more convenient for routine diagnosis of the fragile X syndrome. This method is based on the use of the Expand Long PCR System, which enables the amplification of normal, premutated and full-mutated alleles, and therefore provides complete CGG repeat analysis of the FMR1 gene. Normal alleles were easily detected by ethidium bromide staining of the agarose gels, suggesting that this assay could be used as a screening test for a large number of referrals. The amplified premutations and full mutations were identified by hybridization with a digoxigenin-labeled 5'-(CGG)_5–3' probe, followed by chemiluminescent detection. The accuracy of our Expand Long PCR protocol was confirmed by Southern blot analysis, illustrating that the Expand Long PCR results concur with those of Southern blotting. In this paper we propose a new strategy for molecular diagnosis of the fragile X syndrome in which our Expand Long PCR assay is used as the first screening test for fragile X mutation detection.     

Nonrandom X inactivation and selection of fragile X full mutation in fetal fibroblasts

In the fragile X female carriers the degree of cognitive impairment appears to be correlated with activation status of the X chromosome bearing the expanded trinucleotide repeat in the promoter of the FMR1 gene. In this study we asked if the deviations from the primarily random pattern of X inactivation are related to the selection which is thought to occur against cells carrying the fragile X full mutation (FM) on the active X chromosome. A fibroblast culture derived from a 20-week FM female fetus was serially passaged. The activation ratio (AR) of the culture increased from 0.68 to 0.92 between passages 2 and 9. All higher passage cells (up to 34 passages) display an AR of 1.0, indicating complete absence of cells in which the normal X chromosome would be inactivated. Of 29 clones established from the fetal culture with AR of 0.8, 28 had no visible 5.2-kb band on Southern blots indicating that these 28 clones consisted entirely of cells with FM on their inactive X chromosome. Only a single clone carried the FM on its active X chromosome. The figure of 1 of 29 is much lower than our expectation based on the AR of mass culture. Therefore cloning and serial cultivation indicate the possibility of selection depending on the activation status of the expanded X chromosome in fetal FM female fibroblasts. Am. J. Med. Genet. 86:162–164, 1999. © 1999 Wiley-Liss, Inc.     

The fragile (X) syndrome: the mutation problem

In an attempt to understand the nature of the mutational event leading to the fra(X) syndrome, we have searched for sporadic cases in 3 populations: affected males, affected females, and non-affected transmitting females. In all 3 populations there was a dearth of isolated cases, and the reasons for this are discussed.     

Insert size and flanking haplotype in fragile X and normal populations: possible multiple origins for the fragile X mutation

A number of recent studies have found non-random associationbetween the fragile X mutation and genotypes for the closest-linkedflanking markers, suggesting either a limited number of ‘founder’mutations or, alternatively, a predisposing haplotype for thefragile X expansions. Using three microsatellite markers within150 kb of FRAXA, we have compared haplotypes in a series offragile X males and in a control population and find a markedlydifferent distribution in the two samples, with apparently greaterhaplotype diversity in the fragile X sample. In the controlsample, various non-random associations of CGG repeat numberswith flanking haplotypes were recorded which provide a clueto the likely origins of the fraglle X mutation, suggestingmore than one mechanism for the initial expansion event.     

Affected sibs with fragde X syndrome exhibit an age-dependent decrease in the size of the fragile X full mutation

The sizes of the fragile X mutation in 33 sib pairs affected with fragile X syndrome were determined by Southern blot analysis. An age-dependent decrease in the size of the mutation was found, suggesting positive selection of blood cells carrying small mutations during life or maternal imprinting.     

Short-term memory and cognitive variability in adult fragile X females

We investigated the possibility that adult fragile X [fra(X)] heterozygotes have a distinct or specific cognitive profile, with a particular focus on visuospatial and/or memory deficits. With a sample of 13 adult fra(X) female carriers (2 fra(X) positive) and age-matched control women, we performed 2 tests: Wechsler Adult Intelligence Scale-Revised (WAIS-R) and the Revised Visual Retention Test (RVRT). An identifiable cognitive profile was not evident in the study group, but significant differences were evident in RVRT performance in number correct and number of errors when compared to controls and normative data. The combination of the WAIS-R and RVRT data suggests that the short-term memory component of the tasks may be of more significance than visuospatial performance in the deficits observed.     

A cryptic full mutation in a male with a classical fragile X phenotype

Fragile X syndrome (FRX) is the most common inherited cause of mental retardation affecting approximately 1/4000 males and half as many females. Mosaicism has been reported in 12-41% of male cases. We present a 47-year-old male with the typical FRX phenotype referred for an evaluation of mental retardation and a psychiatric disorder. Analysis of the FMR-1 CGG repeat size was performed on peripheral blood by PCR and Southern blot analysis. The proband was shown to carry a premutation allele of 58 CGG repeats. Because of the compelling clinical phenotype, further testing was performed on DNA extracted from skin fibroblasts, which yielded a 500 CGG repeat allele. Mosaic cases of FRX have been reported but rarely without detectable mosaicism in peripheral blood. Therefore, this case is atypical because of the striking differences in the results obtained for the two different cell types. We concur with others that testing of ectodermally derived tissues may provide improved diagnosis and perhaps better insight into the overall prognosis of the affected individual. This case demonstrates the need to consider further study on other tissues when there is a strong clinical suspicion of FRX.