Fragile X Mental Retardation 1 CGG Repeat Research Articles

Genome-wide screening for genes involved in the epigenetic basis of fragile X syndrome.

SummaryFragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. The epigenetic factors responsible for FMR1 inactivation are largely unknown. Here, we initially demonstrated the feasibility of FMR1 reactivation by targeting a single epigenetic factor, DNMT1. Next, we established a model system for FMR1 silencing using a construct containing the FXS-related mutation upstream to a reporter gene. This construct was methylated in vitro and introduced into a genome-wide loss-of-function (LOF) library established in haploid human pluripotent stem cells (PSCs), allowing the identification of genes whose functional loss reversed the methylation-induced silencing of the FMR1 reporter. Selected candidate genes were further analyzed in haploid- and FXS-patient-derived PSCs, highlighting the epigenetic and metabolic pathways involved in FMR1 regulation. Our work sheds light on the mechanisms responsible for CGG-expansion-mediated FMR1 inactivation and offers novel targets for therapeutic FMR1 reactivation.

Triplet-Repeat Primed PCR and Capillary Electrophoresis for Characterizing the Fragile X Mental Retardation 1 CGG Repeat Hyperexpansions.

Fragile X mental retardation 1 (FMR1) CGG repeat expansions cause fragile X syndrome-the leading monogenic form of intellectual disability-and increase the risk for fragile X-associated tremor ataxia syndrome and fragile X-associated primary ovarian insufficiency. Southern blot (SB) analysis is the current gold standard test for FMR1 molecular diagnosis. Several polymerase chain reaction (PCR)-based methods are now available for sizing FMR1 CGG repeat expansions. These methods offer higher diagnostic sensitivity and specificity compared to SB analysis, significantly reduce the turnaround time and increase throughput. In this chapter, we describe a triplet-repeat primed PCR protocol that employs capillary electrophoresis to resolve the derived amplicon products, enabling precise determination of the FMR1 genotypes in both males and females and characterization of the CGG repeat structure.

ASFMR1 splice variant: A predictor of fragile X-associated tremor/ataxia syndrome.

ObjectiveTo explore the association of a splice variant of the antisense fragile X mental retardation 1 (ASFMR1) gene, loss of fragile X mental retardation 1 (FMR1) AGG interspersions and FMR1 CGG repeat size with manifestation, and severity of clinical symptoms of fragile X-associated tremor/ataxia syndrome (FXTAS).MethodsPremutation carriers (PMCs) with FXTAS, without FXTAS, and normal controls (NCs) had a neurologic evaluation and collection of skin and blood samples. Expression of ASFMR1 transcript/splice variant 2 (ASFMR1-TV2), nonspliced ASFMR1, total ASFMR1, and FMR1 messenger RNA were quantified and compared using analysis of variance. Least absolute shrinkage and selection operator (LASSO) logistic regression and receiver operating characteristic analyses were performed.ResultsPremutation men and women both with and without FXTAS had higher ASFMR1-TV2 levels compared with NC men and women (n = 135,135, p < 0.0001), and ASFMR1-TV2 had good discriminating power for FXTAS compared with NCs but not for FXTAS from PMC. After adjusting for age, loss of AGG, larger CGG repeat size (in men), and elevated ASFMR1-TV2 level (in women) were strongly associated with FXTAS compared with NC and PMC (combined).ConclusionsThis study found elevated levels of ASFMR1-TV2 and loss of AGG interruptions in both men and women with FXTAS. Future studies will be needed to determine whether these variables can provide useful diagnostic or predictive information.

FMR1 CGG repeat expansion mutation detection and linked haplotype analysis for reliable and accurate preimplantation genetic diagnosis of fragile X syndrome.

Fragile X mental retardation 1 (FMR1) full-mutation expansion causes fragile X syndrome. Trans-generational fragile X syndrome transmission can be avoided by preimplantation genetic diagnosis (PGD). We describe a robust PGD strategy that can be applied to virtually any couple at risk of transmitting fragile X syndrome. This novel strategy utilises whole-genome amplification, followed by triplet-primed polymerase chain reaction (TP-PCR) for robust detection of expanded FMR1 alleles, in parallel with linked multi-marker haplotype analysis of 13 highly polymorphic microsatellite markers located within 1 Mb of the FMR1 CGG repeat, and the AMELX/Y dimorphism for gender identification. The assay was optimised and validated on single lymphoblasts isolated from fragile X reference cell lines, and applied to a simulated PGD case and a clinical in vitro fertilisation (IVF)-PGD case. In the simulated PGD case, definitive diagnosis of the expected results was achieved for all 'embryos'. In the clinical IVF-PGD case, delivery of a healthy baby girl was achieved after transfer of an expansion-negative blastocyst. FMR1 TP-PCR reliably detects presence of expansion mutations and obviates reliance on informative normal alleles for determining expansion status in female embryos. Together with multi-marker haplotyping and gender determination, misdiagnosis and diagnostic ambiguity due to allele dropout is minimised, and couple-specific assay customisation can be avoided.

Distribution of fragile X mental retardation 1 CGG repeat and flanking haplotypes in a large Chinese population.

Fragile X syndrome is mainly caused by a CGG repeat expansion within the 5′ UTR of the fragile X mental retardation 1 (FMR1) gene. Previous analyses of the FMR1 CGG repeat patterns and flanking haplotypes in Caucasians and African Americans have identified several factors that may influence repeat instability. However, the CGG repeat patterns and distribution for FRAXAC2 have not yet been investigated in mainland Chinese. We surveyed the CGG repeat lengths in 1113 Han Chinese (534 males and 579 females), and the CGG repeat patterns of 534 males were determined by sequence analysis. We also explored the flanking haplotypes (DXS548-FRAXAC1-FRAXAC2) in 566 unaffected and 28 unrelated fragile X Chinese males. The most frequent alleles for DXS548 and FRAXAC1 were identical between our Chinese population and other Asian populations. We identified several low-abundance alleles for DXS548 and FRAXAC1 not found in previous studies in mainland Chinese and Taiwanese cohorts. The most frequent allele was (CGG)29 followed by (CGG)30, and the most frequent patterns were 9 + 9 + 9, 10 + 9 + 9, and 9 + 9 + 6 + 9, similar to those in Singaporeans. We identified only one premutation female carrier with 89 CGG repeats in the 1113 Han Chinese. A few associations between the CGG repeat patterns and flanking haplotypes were determined in this study. In general, the Chinese population had a smaller number of alleles and lower expected heterozygosity for all three STR markers and FRAXA locus when compared with Caucasians and African Americans. We identified a novel haplotype 7-3-5 + that is significantly associated with the full mutation.

The Significance of Fragile X Mental Retardation Gene 1 CGG Repeat Sizes in the Normal and Intermediate Range in Women With Primary Ovarian Insufficiency

Primary ovarian insufficiency (POI) refers to the cessation of menses before the age of 40 years and occurs in 1% to 2% of women. The specific cause of POI is unknown in the majority of cases. Known causes include gene mutations of fragile X mental retardation 1 (FMR1), a gene located on the X chromosome. This gene codes for a protein essential for normal cognitive development and female reproductive function. FMR1 contains a CCG trinucleotide segment in an untranslated region of its DNA that is repeated less than 45 times in normal individuals (normal range of repeats). Mutation of FMR1 results in abnormal expansion of an unstable CGG triplet, leading to impaired cognitive and reproductive function. Between 55 and 200 CCG repeats is called premutation; affected individuals with premutation are at risk for POI and further expansion of repeats in subsequent generations. People with 45 to 54 CCG repeats (intermediate range of repeats) are considered to be at borderline risk for POI and further expansion of repeats. This case-control study was designed to determine whether the risk of POI is associated with CGG repeat length of normal and intermediate range (up to 55 repeats) in a large cohort of Dutch women with idiopathic POI. The second aim of the study was to examine a possible association between the number of CGG repeats and age at first manifestation of POI as a measurement for the severity of POI. The study population was composed of 375 well-phenotyped Dutch women diagnosed with POI; control subjects were 3368 women with natural menopause at 40 years or older. The FMR1 CGG repeat number was determined by polymerase chain reaction amplification of DNA extracted from blood samples drawn from each patient. Fisher exact test was used to assess the prevalence of intermediate-size CGG repeats in POI cases and control subjects. Analysis of variance tested differences in mean CGG repeat lengths on alleles 1 and 2 between POI cases and control subjects. Allele 1 has the lowest triple repeat number, and allele 2 has the highest triple repeat number. There was no significant difference between POI cases and control subjects in the frequency of intermediate-size CGG repeats on allele 2 (POI 2.7 vs control subjects 3.8%; the odds ratio was 0.72, with a 95% confidence interval of 0.38–1.39; P = 0.38). Linear regression analysis showed no association between the number of CGG repeats and age at first POI manifestation (β = 20.019, P = 0.72). These data suggest that intermediate-size CGG repeats are not associated with risk of POI. Therefore, evaluation of normal and intermediate FMR1 repeat size appears to be of little or no value in the diagnostic workup of women affected by POI or in the prognostic assessment of women at risk of developing POI. Reasons for caution in interpreting these results are as follows: (1) FMR1 CGG repeat lengths in POI cases and control subjects were genotyped in 2 different laboratories; technical differences could have affected the results; (2) distributions of CGG repeats could vary among ethnic populations; (3) women with primary amenorrhea (n = 17) were included in the POI group. The last 2 possible limitations were unlikely because exclusion of data from nonwhite women or women with primary amenorrhea did not affect the results.

Low-normal FMR1 CGG repeat length: phenotypic associations.

This population-based study investigates genotype–phenotype correlations of “low- normal” CGG repeats in the fragile X mental retardation 1 (FMR1) gene. FMR1 plays an important role in brain development and function, and encodes FMRP (fragile X mental retardation protein), an RNA-binding protein that regulates protein synthesis impacting activity-dependent synaptic development and plasticity. Most past research has focused on CGG premutation expansions (41–200 CGG repeats) and on fragile X syndrome (200+ CGG repeats), with considerably less attention on the other end of the spectrum of CGG repeats. Using existing data, older adults with 23 or fewer CGG repeats (2 SDs below the mean) were compared with age-peers who have normal numbers of CGGs (24–40) with respect to cognition, mental health, cancer, and having children with disabilities. Men (n = 341 with an allele in the low-normal range) and women (n = 46 with two low-normal alleles) had significantly more difficulty with their memory and ability to solve day to day problems. Women with both FMR1 alleles in the low-normal category had significantly elevated odds of feeling that they need to drink more to get the same effect as in the past. These women also had two and one-half times the odds of having had breast cancer and four times the odds of uterine cancer. Men and women with low-normal CGGs had higher odds of having a child with a disability, either a developmental disability or a mental health condition. These findings are in line with the hypothesis that there is a need for tight neuronal homeostatic control mechanisms for optimal cognitive and behavioral functioning, and more generally that low numbers as well as high numbers of CGG repeats may be problematic for health.

AGG interruptions and maternal age affect FMR1 CGG repeat allele stability during transmission.

BackgroundThe presence of AGG interruptions in the CGG repeat locus of the fragile X mental retardation 1 (FMR1) gene decreases the instability of the allele during transmission from parent to child, and decreases the risk of expansion of a premutation allele to a full mutation allele (the predominant cause of fragile X syndrome) during maternal transmission.MethodsTo strengthen recent findings on the utility of AGG interruptions in predicting instability or expansion to a full mutation of FMR1 CGG repeat alleles, we assessed the outcomes of 108 intermediate (also named gray zone) and 710 premutation alleles that were transmitted from parent to child, and collected from four international clinical sites. We have used the results to revise our initial model that predicted the risk of a maternal premutation allele expanding to a full mutation during transmission and to test the effect of AGG interruptions on the magnitude of expanded allele instability of intermediate or premutation alleles that did not expand to a full mutation.ResultsConsistent with previous studies, the number of AGG triplets that interrupts the CGG repeat locus was found to influence the risk of allele instability, including expansion to a full mutation. The total length of the CGG repeat allele remains the best predictor of instability or expansion to a full mutation, but the number of AGG interruptions and, to a much lesser degree, maternal age are also factors when considering the risk of transmission of the premutation allele to a full mutation.ConclusionsOur findings demonstrate that a model with total CGG length, number of AGG interruptions, and maternal age is recommended for calculating the risk of expansion to a full mutation during maternal transmission. Taken together, the results of this study provide relevant information for the genetic counseling of female premutation carriers, and improve the current predictive models which calculate risk of expansion to a full mutation using only total CGG repeat length.

Immune Dysregulation as a Cause of Autoinflammation in Fragile X Premutation Carriers: Link between FMRI CGG Repeat Number and Decreased Cytokine Responses

BackgroundIncreased rates of autoinflammatory and autoimmune disorders have been observed in female premutation carriers of CGG repeat expansion alleles of between 55–200 repeats in the fragile X mental retardation 1 (FMR1) gene. To determine whether an abnormal immune profile was present at a cellular level that may predispose female carriers to autoinflammatory conditions, we investigated dynamic cytokine production following stimulation of blood cells. In addition, splenocyte responses were examined in an FMR1 CGG knock-in mouse model of the fragile X premutation.MethodsHuman monocyte and peripheral blood leukocytes (PBLs) were isolated from the blood of 36 female FMR1 premutation carriers and 15 age-matched controls. Cells were cultured with media alone, LPS or PHA. In the animal model, splenocytes were isolated from 32 CGG knock-in mice and 32 wild type littermates. Splenocytes were cultured with media alone or LPS or PMA/Ionomycin. Concentrations of cytokines (GM-CSF, IL-1β, IL-6, IL-10, IL-13, IL-17, IFNγ, TNFα, and MCP-1) were determined from the supernatants of cellular cultures via Luminex multiplex assay. Additionally, phenotypic cellular markers were assessed on cells isolated from human subjects via flow cytometry.ResultsWe found decreases in cytokine production in human premutation carriers as well as in the FMR1 knock-in mice when compared with controls. Levels of cytokines were found to be associated with CGG repeat length in both human and mouse. Furthermore, T cells from human premutation carriers showed decreases in cell surface markers of activation when compared with controls.ConclusionsIn this study, FMR1 CGG repeat expansions are associated with decreased immune responses and immune dysregulation in both humans and mice. Deficits in immune responses in female premutation carriers may lead to increased susceptibility to autoimmunity and further research is warranted to determine the link between FMR1 CGG repeat lengths and onset of autoinflammatory conditions.

Maternal FMR1 premutation allele expansion and contraction in fraternal twins

Fragile X syndrome results from an expansion of the CGG trinucleotide repeat in the 5' untranslated region of the Fragile X Mental Retardation 1 (FMR1) gene. Expansion of a maternal premutation allele is the mechanism by which a full mutation allele arises; contraction of a maternal premutation allele is rare. Here we report on both an expansion and contraction of a maternal FMR1 premutation allele in fraternal twins. The propositus was the product of a 29-week gestation twin pregnancy and was referred for FMR1 testing due to developmental delay. A FMR1 full mutation with complete methylation was observed on Southern blot analysis. Evaluation of the maternal FMR1 gene by PCR revealed a normal and premutation allele with CGG repeat numbers of 30 and 93, respectively. Subsequent FMR1 testing on the twin sister of the propositus detected CGG repeat numbers of 30 and 54. The FMR1 CGG repeat number of the reproductive partner was 30. The FMR1 CGG repeat 30 allele in the twin sister was determined to be of paternal origin and the FMR1 allele with a CGG repeat number of 54 was of maternal origin. This observation is particularly interesting not only because of the concomitant donation of a FMR1 expanded and contracted premutation allele in a twin pregnancy but also because of the significant degree of contraction (39 repeats) of the maternal premutation allele.

Screening for CGG Repeat Expansion in the FMR1 Gene by Melting Curve Analysis of Combined 5′ and 3′ Direct Triplet-Primed PCRs

CGG repeat expansions in the FMR1 (fragile X mental retardation 1) gene are associated with fragile X syndrome, fragile X-associated tremor/ataxia syndrome, and fragile X-associated primary ovarian insufficiency. We evaluated the use of melting curve analysis (MCA) of triplet-primed PCR (TP-PCR) assays as a rapid screening tool for the positive identification of expanded FMR1 alleles in men and women. Both 5'- and 3'-weighted direct TP-PCRs (dTP-PCRs) were evaluated on 29 cell line-derived DNA samples and 44 blinded clinical samples. The presence of expansions was identified by the melting curve profiles generated automatically through MCA on the LightCycler 480 Real-Time PCR System. All samples were also analyzed by capillary electrophoresis to confirm the identities of the PCR fragments that gave rise to the observed melt peak profiles. The presence of expanded alleles in samples from both males and females produced melt peak profiles that were distinct from those of individuals with the normal allelic form. In the blinded test, positive and negative calls for the presence of an expanded allele corroborated with previously determined genotype classifications for all samples. The approach of dTP-PCR plus MCA offers a single-step strategy with high diagnostic sensitivity and specificity for rapid screening detection of FMR1 CGG repeat expansions, regardless of sex. The combined use of 5'- and 3'-weighted dTP-PCR assays minimizes the incidence of false-negative results arising from repeat-flanking deletions.

Methodology Multiple displacement amplification for preimplantation genetic diagnosis of fragile X syndrome

Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples that have genetic risks. Despite the many advantages provided by PGD, there are several problems, including amplification failure, allele drop-out and amplification inefficiency. We evaluated multiple displacement amplification (MDA) for PGD of the fragile X syndrome. Whole genome amplification was performed using MDA. MDA products were subjected to fluorescent PCR of fragile X mental retardation-1 (FMR1) CGG repeats, amelogenin and two polymorphic markers. In the pre-clinical tests, the amplification rates of the FMR1 CGG repeat, DXS1215 and FRAXAC1 were 84.2, 87.5 and 75.0%, respectively, while the allele dropout rates were 31.3, 57.1 and 50.0%, respectively. In two PGD treatment cycles, 20 embryos among 30 embryos were successfully diagnosed as 10 normal embryos, four mutated embryos and six heterozygous carriers. Three healthy embryos were transferred to the uterus; however, no clinical pregnancy was achieved. Our data indicate that MDA and fluorescent PCR with four loci can be successfully applied to PGD for fragile X syndrome. Advanced methods for amplification of minuscule amounts of DNA could improve the sensitivity and reliability of PGD for complicated single gene disorders.

Developmental Study of Fragile X Syndrome Using Human Embryonic Stem Cells Derived from Preimplantation Genetically Diagnosed Embryos

We report on the establishment of a human embryonic stem cell (HESC) line from a preimplantation fragile X-affected embryo and demonstrate its value as an appropriate model to study developmentally regulated events that are involved in the pathogenesis of this disorder. Fragile X syndrome results from FMR1 gene inactivation due to a CGG expansion at the 5'UTR region of the gene. Early events in FMR1 silencing have not been fully characterized due to the lack of appropriate animal or cellular models. Here we show that, despite the presence of a full mutation, affected undifferentiated HESCs express FMR1 and are DNA unmethylated. However, epigenetic silencing by DNA methylation and histone modification occurs upon differentiation. Our unique cell system allows the dissection of the sequence by which these epigenetic changes are acquired and illustrates the importance of HESCs in unraveling developmentally regulated mechanisms associated with human genetic disorders.

Transcript levels of the intermediate size or grey zone fragile X mental retardation 1 alleles are raised, and correlate with the number of CGG repeats

Background: Grey zone or intermediate alleles are one of the three recognised classes of the X-linked fragile X mental retardation 1 (FMR1) gene showing intergenerational instability. These classes are defined...