A new PCR assay useful for screening of FRAXE/FMR2 mental impairment among males

FRAXE (FMR2) is a fragile site associated with mental impairment located in Xq28, 600 kb distal to FRAXA (FMR1), the fragile X syndrome fragile site. The FRAXE mutation is an expansion of a CCG repeat that results in methylation of a nearby CpG island. FRAXE alleles could be divided into four categories: normal (6–30 CCG repeats), intermediate (31–60 CCG repeats), premutation (61–200 CCG repeats), and full mutation (over 200 repeats). We have developed a non‐isotopic polymerase chain reaction (PCR)‐based assay for the identification of FRAXE full mutation alleles among mentally impaired men. In this novel PCR test for the FRAXE locus, we used three primers to permit an amplification of a 223 bp monomorphic internal control fragment in addition to the amplification of a 419 bp (CCG)₁₆ FRAXE locus band. A linear series of 93 male patients referred for FRAXE testing but found to be negative for the (CCG)_n expansion in the FMR2 gene by Southern blotting analysis were retested by our PCR technique. In addition, we analyzed two positive controls consisting of a FRAXE fully mutated male and one male with a Xq terminal deletion. The developed PCR test showed accuracy of 100% in the normal individuals retested by PCR analysis, as well as in the two positive control samples utilized, in which the strategy of multiplex amplification worked as expected. Although not suitable for medical diagnosis of females and mosaics, it constitutes an important strategy for PCR typing and for FRAXE population screening. Hum Mutat 18:157–162, 2001. © 2001 Wiley‐Liss, Inc.     

Fragile-X syndrome and skewed X-chromosome inactivation within a family: a female member with complete inactivation of the functional X chromosome

Fragile X syndrome is the most common form of inherited mental retardation. It is caused by the increase in length of a stretch of CGG triplet repeats within the FMR1 gene. A full mutation (> 200 repeats) leads to methylation of the CpG island and silencing of the FMR1 gene. We present here two sisters that are compound heterozygotes for a full mutation and a 53 repeat intermediate allele, one of them showing mental retardation and clinical features of an affected male (speech delay, hyperactivity, large ears, prominent jaw, gaze aversion), while the other is borderline normal (mild delay). Southern blot and FMRP expression analysis showed that the sister with mental retardation had the normal FMR1 gene totally methylated and no detectable protein, while her sister had 70% of her cells with the normal FMR1 gene unmethylated and normal FMRP levels. We found that the observed phenotypic differences between both sisters who are cytogenetically normal, are caused by extreme skewed X-chromosome inactivation. Analysis of the extended family showed that most of the other female family members that carry a pre-mutation or a full mutation showed some degree of skewing in their X-chromosome inactivation. The presence of several family members with skewed X inactivation and the direction and degree of skewing is inconsistent with a mere selection during development, and suggests a genetic origin for this phenomenon.     

FRAXE mutation in mentally retarded patients using the OxE18 probe

The folate-sensitive fragile site FRAXE is located in proximal Xq28 of the human X chromosome and lies approximately 600 kb distal to the fragile X syndrome (FRAXA) fragile site at Xq27.3. Although FRAXA and FRAXE are indistinguishable by means of conventional cytogenetics, they can now be delineated at the molecular level and provides the basis for a proper diagnosis. The screening for CGG amplifications in the FMR1 gene was based on standard protocols using EcoRI digests on Southern blots and hybridization with the StB12.3 probe. The FRAXE mutation was analyzed by digestion with HindIII and the filters were probed with OxE20. We present the results of 144 patients referred for fragile X testing but negative for the FMR1 gene trinucleotide expansion, that were also screened for the FMR2 expansion. For FRAXE mutation a molecular protocol for OxE18 probe was used, in the DNA samples digested with EcoRI on the same blots as those used for detection of FRAXA. None of the patients tested were positive for the FRAXE expansion. This technique was successfully established into our laboratory routine showing the practical use of testing for FRAXA and FRAXE in a large series of patients.     

A simple multiplex FRAXA, FRAXE, and FRAXF PCR assay convenient for wide screening programs

FRAXA, FRAXE, and FRAXF are folate-sensitive fragile sites originally discovered in patients with X-linked mental retardation. The FMR1 gene, whose first exon includes the FRAXA site on Xq27.3, accounts for 15-20% of all X-linked forms of mental retardation. Loss of expression of FMR2, a gene adjacent to the FRAXE site on Xq28, is correlated with FRAXE expansion in some mild mentally retarded patients. FRAXF is a fragile site whose expression has not been associated with any pathological phenotype. The fragility in all three sites is caused by expansions of CGG repeats adjacent to hypermethylated CpG islands. The prevalence of FRAXA, FRAXE, and FRAXF remains uncertain because of the lack of a simple and cost-effective test allowing wide screening programs. For the same reason, the real phenotype-genotype correlations in FRAXE and FRAXF are uncertain as well. We have developed a rapid multiplex polymerase chain reaction (PCR) assay in which hypermethylated CpG islands adjacent to FRAXA, FRAXE, and FRAXF are displayed. The test is very simple and cost-effective, requires only 30 microl of peripheral blood, and can be used for performing diagnoses, postnatal and prenatal, and for screening large groups of control and mentally retarded males and newborn boys.     

Implication of screening for FMR1 and FMR2 gene mutation in individuals with nonspecific mental retardation in Taiwan.

Fragile X syndrome (FXS) is the most common form of familial mental retardation (MR), attributable to (CGG)n expansion in the FMR1 gene. FRAXE is less frequent, associated with a similar mutation of the FMR2 gene. This study attempted to ascertain the prevalence of both disorders in Taiwan, as well as to develop a method to effectively find carriers. A total of 321 patients with nonspecific MR were screened for the FMR1 and FMR2 mutation. Four of 206 boys and men (1.9%) and 1 in 115 girls and women (0.9%) were identified as having FXS. All four FXS boys or men could be identified by Southern blot analysis, as well as by a simple nonradioactive polymerase chain reaction analysis. None of the 206 boys or men had FMR2 full mutation. This confirmed the low incidence of FRAXE in Chinese. FXS appears to be more prevalent among patients with mild MR, because 4 of the 5 patients with FXS were from the 115 with mild MR (3.48%) and only 1 was from the other 206 with severe MR (0.49%). All five FXS cases were maternally inherited. Other family members were resistant to further searching for carriers. It is worth noting that none of these mothers had a discernible premarital family history of MR. Thus the negative family history could not preclude the possibility that a woman was a carrier. To identify female carriers of childbearing age, beyond the scope of family history, is thus worthy of further exploration. Screening men for carriers using this inexpensive method is probably feasible, even though normal transmitting men have no immediate risk of producing a child with the disease. Female carriers can then be effectively identified from these normal transmitting men and can take all preventive measures.     

Instability of the FMR2 trinucleotide repeat region associated with expanded FMR1 alleles

The fragile sites FRAXA and FRAXE, located ∼600 kb apart on Xq27.3 and Xq28, respectively, are due to a CGG trinucleotide repeat expansion. Although the expansion mechanism for these and other trinucleotide repeat disorders remains unknown, the similarities between the FRAXA and FRAXE regions suggest a possible association between the 2 sites. DNA from 953 individuals was analyzed to determine the distribution of FRAXE repeat sizes in this population and to ascertain potential association between FRAXA and FRAXE repeat sizes. Thirty-four FMR2 alleles ranging from 3–42 repeats were identified. No FRAXE expansions were found in this population, supporting previous findings that FRAXE expansions are rare. However, in the fragile X syndrome affected group, a FMR2 delection, 2 cases of FRAXE repeat instability and a FRAXE mosaic male were identified. Also, a previously identified, rare FMR2 polymorphism was observed. Statistical analyis showed no correlation between normal FRAXA and FRAXE repeat sizes studied, although there was a significant size difference in larger FMR2 alleles that segregated with expanded FMR1 alleles. These findings support the idea of an association between repeat expansion in the FMR1 gene and instability or deletions in the FMR2 gene. Am. J. Med. Genet. 73:447–455, 1997. © 1997 Wiley-Liss, Inc.     

Microdeletion of Xq28 involving the AFF2 (FMR2) gene in two unrelated males with developmental delay

Fragile X E (FRAXE) is an X‐linked form of intellectual disability characterized by mild to moderate cognitive impairment, speech delay, hyperactivity, and autistic behavior. The folate‐sensitive fragile site FRAXE is located in Xq28 approximately 600 kb distal to the fragile X syndrome fragile site (FRAXA) and harbors an unstable GCC (CCG) triplet repeat adjacent to a CpG island in the 5′ untranslated region of the AFF2 (FMR2) gene. The disorder results from amplification and methylation of the GCC repeat and resultant silencing of AFF2. Although chromosome abnormalities that disrupt AFF2 have been reported in two individuals with mild‐moderate intellectual disability, microdeletions of Xq28 that delete only AFF2 have not been described as a potential cause of FRAXE‐intellectual disability. We performed clinical and molecular characterization of two males with 240 and 499 kb deletions, respectively, at Xq28, both of which encompassed only one gene, AFF2. The 240 kb deletion in Patient 1 was intragenic and lead to the loss of 5′ exons 2–4 of AFF2; the 499 kb deletion in Patient 2 removed the 5′ exons 1–2 of AFF2 including approximately 350 kb upstream of the gene. Both individuals had developmental and speech delay, and one had mild dysmorphism. We predict disruption of AFF2 in these two patients is likely the cause of their overlapping phenotypes. © 2011 Wiley Periodicals, Inc.     

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.     

Fragile X founder effects and new mutations in Finland

The apparent associations between fragile X mutations and nearby microsatellites may reflect both founder effects and microsatellite instability. To gain further insight into their relative contributions, we typed a sample of 56 unrelated control and 37 fragile X chromosomes from an eastern Finnish population for FMR1 CGG repeat lengths, AGG interspersion patterns, DXS548, FRAXAC1, FRAXE and a new polymorphic locus, Alu-L. In the controls, the most common FMR1 allele was 30 repeats with a range of 20 to 47 and a calculated heterozygosity of 88%. A strong founder effect was observed for locus DXS548 with 95% of fragile X chromosomes having the 21 CA repeat (196 bp) allele compared to 17% of controls, while none of the fragile X but 69% of controls had the 20 repeat allele. Although the FRAXAC1 locus is much closer than DXS548 to FMR1 (7 kb vs. 150 kb), there was no significant difference between fragile X and control FRAXAC1 allele distributions. The FRAXE repeat, located 600 kb distal to FMR1, was found to show strong linkage disequilibrium as well. A newly defined polymorphism, Alu-L, located at ∼40 kb distal to the FMR1 repeat, showed very low polymorphism in the Finnish samples. Analysis of the combined loci DXS548-FRAXAC1- FRAXE showed three founder haplotypes. Haplotype 21-19-16 was found on 27 (75%) of fragile X chromosomes but on none of controls. Three (8.4%) fragile X chromosomes had haplotypes 21-19-15, 21-19-20, and 21-19-25 differing from the common fragile X haplotype only in FRAXE. These could have arisen by recombination or from mutations of FRAXE. A second haplotype 21-18-17 was found in four (11.1%) fragile X chromosomes but only one (1.9%) control. This may represent a more recent founder mutation. A third haplotype 25-21-15, seen in two fragile X chromosomes (5.6%) and one (1.9%) control, was even less common and thus may represent an even more recent mutation or admixture of immigrant types. Analysis of the AGG interspersions within the FMR1 CGG repeat showed that 7/8 premutation chromosomes lacked an AGG whereas all controls had at least one AGG. This supports the hypothesis that the mutation of AGG to CGG leads to repeat instability and mutational expansion. © 1996 Wiley-Liss, Inc.     

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.     

Effect of fragile X status categories and FMRP deficits on cognitive profiles estimated by robust pedigree analysis

The effect of the fragile X pre-mutation and full mutation categories, and FMRP deficits in these categories, on neurocognitive status, have been assessed in fragile X individuals from 144 extended families, which included fragile X individuals, as well as their non-fragile X relatives. Neuropsychological status was assessed by the Wechsler summary and subtest test scores. A modification of the maximum likelihood estimators for pedigree data that is resistant to outliers was used to analyze the data. The results have demonstrated the effect of large expansions of CGG repeat in the FMR1 (fragile X mental retardation 1) gene (full mutation) in decreasing full scale IQ (FSIQ), as well as several FSIQ-adjusted subtest scores in the performance domain. Moreover, the results have demonstrated significant cognitive deficits in male individuals with pre-mutation. FMRP depletion correlates strongly with neurocognitive status in the full mutation subjects. Evidence for the effect of FMRP in smaller expansions (pre-mutation) in reducing FSIQ, Performance and Verbal scores, as well as subtest scores in males, has also been obtained. The results are also suggestive of factors other than FMRP deficit which may determine some specific cognitive deficits in fragile X pre-mutation carriers. Genetic variance estimated from the models accounts for less than half of the total variance in FSIQ, and it varies widely between individual Wechsler subtests.