Folate metabolism in cells from fragile X syndrome patients and carriers

The in vitro folate sensitivity of the fragile site at Xq27 and the claims of a beneficial response of patients given folic acid prompted us to examine the folate metabolism in cells cultured from fragile X syndrome patients and carriers. Using Epstein-Barr virus we established permanent lymphoblastoid lines from 4 fragile X syndrome males and 3 carriers from 7 families. All these lines expressed the fragile site when 0.1 microM 5-fluorodeoxyuridine (FUdR) was added to the cultures 24 hr prior to harvest; thus, the lines seemed suitable for seeking an intrinsic defect. Fragile X syndrome patient and carrier lines and normal control cell lines did not differ in regard to folate requirement for growth, the ability to use homocysteine in place of methionine, the ability to utilize reduced folates as the sole folate source, or methotrexate sensitivity. These results suggest that no intrinsic defect in folate metabolism is present in fragile X syndrome cells.     

The fragile X syndrome: the patients and their chromosomes.

We have reviewed recent publications, mostly from 1980 onwards, concerned with the problem of identifying patients with the fragile X chromosome and mental retardation, considering the two practical sides of the problem, that is, identification by their external appearance and by chromosomal studies. We conclude that this condition covers a large range of physical findings which occur in varying degrees in people with the chromosome marker. We have tried to clarify the existent criteria that have to be considered for an accurate cytogenetic diagnosis.     

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.     

Inheritance of the fragile X syndrome: size of the fragile X premutation is a major determinant of the transition to full mutation.

The fragile X mental retardation syndrome is caused by unstable expansion of a CGG repeat. Two main types of mutation have been categorised. Clinical expression is associated with the presence of the full mutation, while subjects who carry only a premutation do not have mental retardation. Premutations have a high risk of transition to full mutation when transmitted by a female. We have used direct detection of the mutations to characterise large families who illustrate the wide variation in penetrance which has been observed in different sibships (a feature often called the Sherman paradox). A family originally found to show tight genetic linkage between the factor 9 gene and the fragile X locus was reanalysed, confirming the original genotype assignments and the observed linkage. The size of premutations was measured by Southern blotting and by using a PCR based test in 102 carrier mothers and this was correlated with the type of mutation found in their offspring. The risk of transition to full mutation was found to be very low for premutations with a size increase (delta) of about 100 bp, increasing up to 100% when the size of premutation was larger than about 200 bp, even after taking into account (at least partially) ascertainment bias. These results confirm and extend those reported by Fu et al (1991) and Yu et al (1992) and explain the Sherman paradox.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transition from premutation to full mutation in fragile X syndrome is likely to be prezygotic

In the fragile X syndrome, the transition from unmethylated moderate expansions of the CGG repeat (premutations) to methylated large expansions (full mutations) occurs only through maternal transmission. The risk of such transition is highly correlated with the size of the maternal premutation (PM), being very low for small PM alleles (approximately 60 repeats), to 100% for alleles above 100 repeats. The timing of this transition was the object of much speculation. A postzygotic transition was proposed as a preferred model, based on the observation that males with full mutation (FM) have PM in sperm. Analysis of tissues from affected fetuses, including additional data reported here, indicate that such a putative postzygotic transition would have to occur very early in embryogenesis and most likely before determination of germ cell lineage. At least 15% of carriers of a FM show a significant proportion of white blood cells carrying a PM (mutation mosaics). We performed a simulation study showing that, if transition to FM is postzygotic, one should observe a much higher proportion of such mosaics in offspring of mothers with small PMs. This was compared with the actual pattern observed in 212 mutated offspring of 112 PM carrier mothers. We found no effect of maternal PM size on incidence of mosaicism in leucocytes. We propose that this is strong, albeit indirect evidence against a postzygotic transition to FM. A transition at an early morula stage (before day 3) cannot, however, be formally excluded.      

The female and the fragile X. A study of 144 obligate female carriers

In the present report we summarize our data on 144 obligate female carriers. The clinical and cytogenetic data are reviewed and discussed. In more than 30% of heterozygotes mental development is borderline to subnormal, and they may also present psychiatric disturbances of which psychotic behaviour is the most frequent. Partial phenotypic expression is present in 28% of the carriers, and is more frequent in the mentally subnormal. Repeated fragile X screening remains negative in more than 50% of the patients. This results in great difficulties in accurate genetic counseling of the individual female at risk. In all females with partial clinical expression fragile X screening is positive. Fragile X carriers have a high fertility. A fourfold increase in twinning is observed and this may be an indication of a disturbed cortico-hypothalamic-hypophyseal axis.     

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.     

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.     

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.     

An unusual fragile X sibship: female compound heterozygote and male with a partially methylated full mutation

We describe here a fragile X sibship of borderline retarded sister and brother born to carrier parents. The sister is a compound heterozygote (with a full mutation on one X chromosome and a pre-mutation on the other X chromosome). The brother has a partially methylated full mutation. The activation ratio (AR) for the sister's pre-mutation was 0.69 and the percent lack of methylation for the brother's full mutation was 73%. Intellectual and neuropsychological Wechsler Adult Intelligence Scale (W.A.I.S.) achievement tests reported full scale IQ scores of 74 in the sister and 77 in the brother. A significant discrepancy between verbal and performance IQ was found in the sister, indicating that her main impairment was in the cognitive area. The parents of this unusual sibship came from a small village, as did one of the two previously described cases of compound heterozygous females. These rare females raise special issues for genetic counselling in fragile X carrier couples, the frequency of which remains to be defined in different populations.     

The female and the fragile X syndrome: data on clinical and psychological findings in 7 fra(X) carriers

In this report we present precise data on the clinical, intellectual and behavioural findings in 7 young fra(X) positive girls. The two most common and most important findings are an overgrowth syndrome present from birth on and common behavioural features like severe attentional problems and extreme shyness and anxiety. These symptoms seem to constitute the major criteria for fra(X) screening in prepubertal girls. The findings in previous studies are compared with the present observations.     

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.     

Carrier testing in the fragile X syndrome: Attitudes and opinions of obligate carriers

This study surveyed obligate carriers of the fragile X syndrome fra(X) to ascertain opinions and attitudes regarding carrier testing. Female carriers of fra(X) syndrome were recruited during their visits to the Fragile X Clinic at Duke University Medical Center. Twenty-eight obligate carriers completed a 48 question structured interview and a visual analog scale (VAS). Strong trends in the responses were identified. Fra(X) syndrome was viewed as a very serious problem and the risk to offspring high. Subjects reported that prior knowledge of carrier status would have changed their reproductive plans. All felt that relatives should be informed about the inheritance of fra(X) syndrome; the mean age given for preferred age to inform their children of the inheritance of fra(X) syndrome was 12 years, and mean age given for optimal timing of carrier testing was 10 years. The women interviewed indicated that growing up with knowledge of their carrier status would have been preferable to learning this information as adults and they endorsed an aggressive approach to informing and testing their children. Further investigation is warranted to determine the psychological consequences of carrier testing for fra(X) syndrome in order to develop appropriate guidelines for testing and informing individuals at risk for fra(X) syndrome. Am. J. Med. Genet 68:62–69, 1997 © 1997 Wiley-Liss, Inc.     

The fragile X syndrome I: familial variation in the proportion of lymphocytes with the fragile site in males

A review of 61 males with the fragile X positive form of the Martin Bell syndrome from 30 families seen in the past 4 years suggests that the number of lymphocytes with the fragile site on the X chromosome (fra(X) ) in retarded males tends to be characteristic for the individual and similar to that found in other retarded males in the same family. The number of lymphocytes with fra(X) was not correlated with height, weight, occipitofrontal circumference, ear length or mean testicular volume in adults nor with age over the whole series.