Segregation of the fragile X mutation from a male with a full mutation: Unusual somatic instability in the FMR-1 locus

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. © 1996 Wiley-Liss, Inc.     

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.     

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.     

Tissue heterogeneity of the FMR1 mutation in a high-functioning male with fragile X syndrome.

Few studies have been conducted comparing the FMR1 mutation in multiple tissues of individuals affected with fragile X syndrome. We report a postmortem study of the FMR1 mutation in multiple tissues from a high-functioning male with fragile X syndrome. This man was not mentally retarded and had only a few manifestations of the disorder such as learning disabilities and mild attention problems. Southern blot analysis of leukocytes demonstrated an unmethylated mutation with a wide span of sizes extending from the premutation to full mutation range. A similar pattern was seen in most regions of the brain. In contrast, a methylated full mutation of a single size was seen in the parietal lobe and in most non-brain tissues studied. Therefore, there were striking differences in both FMR1 mutation size and methylation status between tissues. Lack of mental retardation in this individual may have been due to sufficient expression of FMR1 protein (FMRP) in most areas of the brain. Immunocytochemistry showed FMRP expression in regions of the brain with the unmethylated mutation (superior temporal cortex, frontal cortex, and hippocampus) and no expression in the region with the methylated full mutation (parietal). Neuroanatomical studies showed no dendritic spine pathology in any regions of the brain analyzed.     

FXTAS in an unmethylated mosaic male with fragile X syndrome from Chile

Carriers of an FMR1 premutation allele (55–200 CGG repeats) often develop the neurodegenerative disorders, fragile X‐associated tremor/ataxia syndrome (FXTAS). Neurological signs of FXTAS, parkinsonism and rapid onset of cognitive decline have not been reported in individuals with an unmethylated full mutation (FM). Here, we report a Chilean family affected with FXS, inherited from a parent carrier of an FMR1 unmethylated full mosaic allele, who presented with a fast progressing FXTAS. This case suggests that the definition of FXTAS may need to be broadened to not only include those with a premutation but also those with an expanded allele in FM range with a lack of methylation leading to elevated FMR1‐mRNA expression levels and subsequent RNA toxicity.     

Neuropsychological dysfunction among affected heterozygous fragile X females.

Fragile X (or Martin-Bell) syndrome is an X-linked disorder that often produces mental retardation in males, but usually affects heterozygous females to a lesser degree. Here we report the results of a brief neuropsychological examination of 20 heterozygous fra(x) girls and women and two control groups of 20 individuals each. One control group was composed of fra(x)-negative mothers (obligate carriers) and sisters of male probands with fra(x) syndrome, whereas the other was composed of 14 head-injured and six learning disabled women and girls. In addition to general intellectual impairment, several specific cognitive deficits were consistently found in individuals with the Martin-Bell syndrome, suggesting focal neuropsychological dysfunction. Significant differences were noted between fra(x) individuals and controls on most cognitive and neuropsychological measures studied. Over one-third of the fra(x) individuals demonstrated neuropsychological symptoms characteristic of the full developmental Gerstmann syndrome, whereas another third had three or four of the five signs of possible parietal lobe dysfunction. In our sample, there was an association between improved performance and increasing age. Differences among heterozygous individuals in number of focal symptoms may reflect some variability in the penetrance of the fra(x) gene, as well as in the functional organization of the brain.     

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.     

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.     

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)     

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.     

Prenatal diagnosis of a hypermethylated full fragile X mutation in chorionic villi of a male fetus.

Fragile X syndrome, one of the most common human genetic diseases, is characterised by a unique genetic mechanism which involves dynamic mutation because of a heritable unstable DNA sequence and abnormal DNA methylation. Direct detection of the dynamic mutation and its methylation status at the DNA level would facilitate reliable tests for prenatal and postnatal diagnosis of the disease and for carrier detection. However, it has been suggested that DNA methylation can not be used as the basis for prenatal diagnosis as the CpG island is not always methylated in chorionic villus DNA. We report here a male fetus exhibiting both extensive somatic heterogeneity and abnormal hypermethylation of the full fragile X mutation in chorionic villus DNA as well as in fetal tissue DNA. Our results indicate that both somatic heterogeneity and hypermethylation of the full fragile X mutation are events that are clearly detectable in the 11th to 12th week of pregnancy.     

Postmortem examination of two fragile X brothers with an FMR1 full mutation.

Large expansions of the CGG repeat in the 5' untranslated region of the FMR1 gene are found in patients with the fragile X syndrome. Amplified CGG repeats in FMR1 are unstable and show intergenerational increase from mother to offspring. The exact timing of repeat amplification, however, is unknown. We have compared the extent of CGG expansion in various tissues of this deceased fragile X patient, and found only limited variation in repeat expansion. The repeat was fully methylated in all tissues examined. Therefore, no evidence for extensive mitotic expansion of the CGG repeat during fetal or postnatal life of a fragile X patient was found, in contrast to dynamic mutations caused by CAG/CTG repeat expansion. Extensive pathological examination of this patient and his affected brother revealed no evidence for specific abnormalities relevant to fragile X syndrome; cerebellar hypoplasia, which has been reported in this disorder, was not evident in either patient.     

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)     

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.     

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.      

Pilot fragile X screening in normal population of Taiwan.

Fragile X syndrome (FXS) is the most common hereditary form of mental retardation. Molecular analysis of the FMR1 gene has now been applied to diagnosis and carrier detection. Because treatment is not feasible, prevention of FXS by prenatal diagnosis of carrier women early during pregnancy is important. The aim of this pilot study was to ascertain the prevalence of mutant FMR1 gene in normal population of Taiwan and to evaluate the efficacy of a betaine-based polymerase chain reaction (PCR) and nonradioactive Southern blot assays. The DNA was randomly and anonymously collected from 100 women and 100 men. The results showed 62% of the women were heterozygous for the CGG-repeat size in FMR1 gene. One of 300 X chromosomes in this study showed premutation, with 95 CGG repeats. All other chromosomes have CGG repeats ranging from 19 to 52, with eight chromosomes (3%) having more than 40 CGG repeats. The most prevalent allele has 29 repeats (48.1%), followed by 30 (24.0%) and 36 (9.5%), respectively. The results of this study reconfirmed previous reports that the prevalent FMR1 CGG repeat alleles in Chinese population are different from that of other populations. However, the prevalence of premutation gene seems to be comparable among them. The betaine-based PCR could minimize the intrinsic problem of preferential amplification and may reliably determine the different allele repeats in heterozygous females. This nonradioactive Southern blot protocol is safe, efficient, and inexpensive. However, further technical improvement may be needed to be more cost-effective for a wide screening of all pregnant women.     

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.