Mosaicism in a fragile X male including a de novo deletion in the FMR1 gene.

In most cases the fragile X syndrome is caused by an amplification of the CGG trinucleotide repeat in the 5' untranslated region of the FMR1 gene, in combination with the hypermethylation of the proximal CpG island. Recently, also a few cases with deletions or a mosaic of a deletion and a full mutation in the FMR1 gene, leading to the same phenotype, have been described. Here we report the molecular analysis of a patient with typical fragile X phenotype and mosaicism of the FMR1 genomic region consisting of a premutation, a full mutation of the CGG repeats, and a 215 bp deletion, diagnosed by Southern blot hybridisation and polymerase chain reaction (PCR). Sequence analysis of the deletion demonstrated that the 5' breakpoint of the deletion is located within a putative hotspot region 75-53 bp proximal to the CGG repeat.     

Mosaicism for an FMR1 gene deletion in a fragile X female

Most cases of fragile X syndrome result from expansion of CGG repeats in the FMR1 gene; deletions and point mutations of FMR1 are much less common. Mosaicism for an FMR1 full mutation with a deletion or with a normal allele has been reported in fragile X males. Here we report on a fragile X female who is mosaic for an FMR1 full mutation and an intragenic deletion. The patient is a 4-year-old girl with developmental delay, autistic-like behaviors, and significant speech and language abnormalities. Southern blotting demonstrated the presence of a methylated full mutation, a normal allele in methylated and unmethylated forms, and an additional fragment smaller than the normal methylated allele. This result indicates that the patient is mosaic for a full mutation and a deletion, in the presence of a normal allele. By DNA sequence analysis, we mapped the 5' breakpoint 63/65 bp upstream from the CGG repeat region and the 3' breakpoint 86/88 bp downstream of the CGG repeats within the FMR1 gene. The deletion removed 210 bp, including the entire CGG repeat region. The full mutation was inherited from a premutation in the patient's mother. The deletion, which remained methylated at the Eag I and Nru I sites, was probably derived from the full mutation allele. Mosaicism of this type is rare in females with a fragile X mutation but should be kept in mind in the interpretation of Southern blots.     

Monozygotic twin brothers with the fragile X syndrome: different CGG repeats and different mental capacities

Little is known about the mechanism of CGG instability and the time frame of instability early in embryonic development in the fragile X syndrome. Discordant monozygotic twin brothers with the fragile X syndrome could give us insight into the time frame of the instability. We describe monochorionic diamniotic twin brothers with the fragile X syndrome who had different CGG repeats and different mental capacities, whereas the normal mother had a premutation. The more retarded brother had a full mutation in all his cells and no FMR-1 protein expression in lymphocytes, whereas the less retarded brother had 50%/50% mosaicism for a premutation and full mutation and FMR-1 protein expression in 26% of his lymphocytes. The differences in repeat size could have arisen either before or after the time of splitting. The time of splitting in this type of twin is around day 6-7. Given the high percentage of mosaicism, we hypothesise that the instability started before the time of splitting at day 6-7.     

Fragile X mutation and FG syndrome-like phenotype

We present data on 4 mentally retarded brothers, 2 of whom were dizygotic twins with congenital hypotonia, constipation, head size disproportionately large for length or height, and a combination of minor anomalies suggestive of FG syndrome. These brothers have a mentally retarded full sister with similar minor anomalies and an older half-brother with the Martin-Bell syndrome. The mother is mentally retarded; 4 of 7 individuals are positive for fragile X, but all have a CGG expansion ranging from 0.2–2 to 4 kb. Although the phenotype is not completely typical of the FG syndrome and the coincidence of the FMR1 mutation and segregation of the MCA/MR phenotype are highly unlikely, the FMR1 mutation may affect morphogenesis more extensively and differently than the Martin-Bell syndrome does to effect an FG syndromelike phenotype in certain families. This phenotype does not appear to be a contiguous gene syndrome, but an effect of the FMR1 mutation on an adjacent gene must be considered. © 1996 Wiley-Liss, Inc.     

Exceptional good cognitive and phenotypic profile in a male carrying a mosaic mutation in the FMR1 gene

Fragile X (FRAX) syndrome is a commonly inherited form of mental retardation resulting from the lack of expression of the fragile X mental retardation protein (FMRP). It is caused by a stretch of CGG repeats within the fragile X gene, which can be unstable in length as it is transmitted from generation to generation. Once the repeat exceeds a threshold length, the FMR1 gene is methylated and no protein is produced resulting in the fragile X phenotype. The consequences of FMRP absence in the mechanisms underlying mental retardation are unknown. We have identified a male patient in a classical FRAX family without the characteristic FRAX phenotype. His intelligence quotient (IQ) is borderline normal despite the presence of a mosaic pattern of a pre-mutation (25%), full mutation (60%) and a deletion (15%) in the FMR1 gene. The cognitive performance was determined at the age of 28 by the Raven test and his IQ was 81. However, FMRP expression studies in both hair roots and lymphocytes, determined at the same time as the IQ test, were within the affected male range. The percentage of conditioned responses after delay eyeblink conditioning was much higher than the average percentage measured in FRAX studies. Moreover, this patient showed no correlation between FMRP expression and phenotype and no correlation between DNA diagnostics and phenotype.     

Paternal retraction of a fragile X allele to normal size,showing normal function over two generations

The FMR1 premutation (PM:55-199 CGG) is associated with fragile X-associated tremor/ataxia syndrome (FXTAS) and when maternally transmitted is at risk of expansion to a hypermethylated full mutation (FM: ≥ 200 CGG) that causes fragile X syndrome (FXS). We describe a maternally transmitted PM (77 CGG) that was passed to a son (103 CGG), and to a daughter (220–1822 CGG), who were affected with FXTAS and FXS, respectively. The male with the PM showed low-level mosaicism for normal size of 30 and 37 CGG. This male had two offspring: one female mosaic for PM and FM (56, 157, >200 CGG) and another with only a 37 CGG allele detected in multiple tissues, neither with a clinical phenotype. The female with the 37 CGG allele showed normal levels of FMR1 methylation and mRNA and passed this 37 CGG allele to one of her daughters, who was also unaffected. These findings show that post-zygotic paternal retraction can lead to low-level mosaicism for normal size alleles, with these normal alleles being functional when passed over two generations.     

Hotspot for deletions in the CGG repeat region of FMR1 in fragile X patients

The fragile X syndrome is the most frequent cause of inheritedmental retardation. The molecular mechanism of the disorderis based on the expansion of a CGG repeat in the 5' UTR of theFMR1 gene In the majority of fragile X patients. The instabilityof this CGG repeat containing region is not restricted to theCGG repeat Itself but expands to the flanking region as well.We describe four unrelated fragile X patients that are mosaicfor both a full mutation and a small deletion in the CGG repeatcontaining region. Sequence analysis of the regions surroundingthe deletions showed that both the (CGG)_n repeat and some flankingsequences were missing in all four patients. The 5' breakpointsof the deletions were found to be located between 75–53bp proximal to the CGG repeat. This suggests the presence ofa hot spot region for deletions in the CGG repeat region ofthe FMR1 gene and emphasizes the instability of this regionIn the presence of an expanded CGG repeat.     

Deletion in the FMR1 gene in a fragile-X male

The pathogenesis of Fragile-X syndrome is a consequence of absence of the FMR1 gene product associated with expansion of the CGG repeat and abnormal methylation of this and a CpG island 250 bp proximal to the CGG repeat located at exon 1 in the FMR1 gene. While this is usually the case, some suspected Fragile-X syndrome patients have been described with a mutation other than CGG expansion. We describe here an affected Fragile-X male, who was found to be mosaic of a full mutation of the CGG expansion and a deletion in the FMR1 gene. The patient's phenotype is probably mainly due to the effect of the full mutation of the repeat sequence. Thus, the influence of the deletion is difficult to evaluate. © 1996 Wiley-Liss, Inc.     

Single-strand conformation polymorphism analysis in the FMR1 gene.

The fragile X syndrome is due to an expansion of the CGG trinucleotide repeat in the FMR1 gene and hypermethylation of its 5' upstream CpG island in about 95% of the cases. The remaining 5% of cases correspond to other molecular alterations in FMR1 gene such as partial or complete deletions, or point mutations within the coding sequence. We selected 31 patients with clinical manifestations of fragile X syndrome, scoring 16 or more in Hagerman's checklist, but without the CGG expansion. We performed single-strand conformation polymorphism analysis using a nonradioactive technique (silver staining) and we detected six anomalous migrations that, by sequence analysis, corresponded to six nucleotide changes. We screened two different populations (control and fragile X) for these changes, and concluded that they correspond to five new polymorphisms within the FMR1 gene and to one possible synonymous mutation.     

A unique case of reversion to normal size of a maternal premutation FMR1 allele in a normal boy

Fragile X syndrome (FXS) is caused mostly by expansion and subsequent methylation of the CGG repeat in the 5'UTR of the FMR1 gene, resulting in silencing of the gene, absence of FMRP and development of the FXS phenotype. The expansion also predisposes the CGG repeat and the flanking regions to further instability that may lead to mosaics between a full mutation and a premutation or, rarely, a normal or deleted allele. Here, we report on a 10-year-old boy with no FXS phenotype, who has a normal CGG tract, although he inherited the maternal expanded allele that causes FXS in his two brothers. Southern blotting demonstrated that the mother carries a premutation allele ( approximately 190 CGG), whereas the propositus shows a normal 5.2 kb fragment after HindIII digestion and a smaller 2.2 kb fragment after double HindIII-EagI digestion, without any apparent mosaicism in peripheral blood leukocytes. PCR and sequence analysis of the FMR1 5'UTR revealed an allele of 43 repeats, with two interspersed AGG triplets in position 10 and 25 and an exceptional CCG triplet in position 17. This latter creates an abnormal EagI site compatible with the smaller 2.2 kb fragment observed with Southern blotting. Haplotype analysis proved that the rearranged allele originated from the maternal expanded allele. To the best of our knowledge, this is the first non-mosaic case of reduction in the CGG tract of the FMR1 gene, resulting in a normal allele.     

Segregation of the fragile X mutation from an affected male to his normal daughter.

We report here a family in which the fragile X mutation segregates from an affected grandfather through his normal daughter to an affected grandson. The grandson shows clinical and cytogenetic expression of fragile X syndrome due to a full mutation (large methylated insertion) in the fragile X gene (FMR-1). The mother shows a premutation (small unmethylated insertion) in her FMR-1 gene as the sole manifestation of the fragile X syndrome. The grandfather expresses the fragile X syndrome at the clinical and cytogenetic level, whereas he is mosaic for a methylated full mutation and an unmethylated premutation. The absence of expression of the fragile X mutation when transmitted through an expressing male might present further evidence for genomic imprinting of the FMR-1 gene. Alternatively, it is possible that the grandfather transmitted his premutation to his daughter due to germline mosaicism with both the premutation and the full mutation present in his sperm.     

Molecular-intelligence correlations in young fragile X males with a mild CGG repeat expansion in the FMR1 gene

Several mechanisms can explain the occurrence of full-mutation fragile X males with an IQ level above −2 SD below mean, also called “high-functioning fragile X males.” Incomplete methylation of the CpG island at the 5′ end of the FMR1 gene is one of these mechanisms. The present study describes the physical and behavior phenotypes in 7 fragile X boys with CGG repeat insertions in the FMR1 gene between 600–2,400 base pairs. The degree of methylation at the FMR1-associated CpG island ranges in peripheral blood lymphocytes from 0–95%. Subjects with a low degree of methylation at this site have mild or absent physical characteristics of the fragile X syndrome, while subjects with a high degree of methylation at this site have more severe physical characteristics. In this range of CGG repeat insertion (600–2,400 base pairs), the degree of methylation at the FMR1-associated CpG island is a good predictor of intelligence, while CGG repeat insertion length is not. © 1996 Wiley-Liss, Inc.