FMR1 Alleles Research Articles

Identification of a novel epigenetic marker for typical and mosaic presentations of Fragile X syndrome

ABSTRACT Background Fragile X syndrome (FXS) is primarily due to CGG repeat expansions in the FMR1 gene. FMR1 alleles are classified as normal (N), intermediate (I), premutation (PM), and full mutation (FM). FXS patients often carry an FM, but size mosaicism can occur. Additionally, loss of methylation boundary upstream of repeats results in de novo methylation spreading to FMR1 promoter in FXS patients. Research design and methods This pilot study investigated the methylation boundary and adjacent regions in 66 males with typical and atypical FXS aged 1 to 30 years (10.86 ± 6.48 years). AmplideX FMR1 mPCR kit was used to discriminate allele profiles and methylation levels. CpG sites were assessed by pyrosequencing. Results 40 out of 66 FXS patients (60.6%) showed an exclusive FM (n = 40), whereas the remaining (n = 26) exhibited size mosaicism [10 PM_FM (15.15%); 10 N_FM (15.15%); 2 N_PM_FM (3%)]. Four patients (6.1%) had deletions near repeats. Noteworthy, a CpG within FMR1 intron 2 displayed hypomethylation in FXS patients and hypermethylation in controls, demonstrating remarkable specificity, sensitivity, and accuracy when a methylation threshold of 69.5% was applied. Conclusions Since intragenic methylation is pivotal in gene regulation, the intronic CpG might be a novel epigenetic biomarker for FXS diagnosis.

P-714 Clinical utility of FMR1 screening: risk factor or monogenic cause for POI

Abstract Study question Is there a risk factor association or a monogenic relationship between FMR1 premutation and developing Fragile X-associated primary ovarian insufficiency (FXPOI)? Summary answer There is an association between FMR1 premutation and FXPOI rather than a monogenic relationship, which is highly dependent on ethnicity. What is known already Among the 40 genes involved in primary ovarian insufficiency (POI), identified in our recent systematic review (Van Der Kelen et al., 2022), the FMR1 premutation is considered as the most common cause of POI. A premutation in the FMR1 gene is defined as a CGG trinucleotide repeat length between 55 and 200 in the 5′ untranslated region. The term FXPOI is used for women with premutation in FMR1 who have a loss of normal function of the ovaries before the age of 40. There is conflicting evidence about the relationship between POI and length of premutation FMR1 alleles. Study design, size, duration Curated publications identified in PubMed and Web of Science on genetics of human female infertility and sex development by using the MESH terms, key words and inclusion/exclusion criteria described in our comprehensive systematic review were subjected to screening for “FMR1 gene.” The articles included cover a period from 1988 to the 1st of November 2021. Participants/materials, setting, methods A total of 161 publications in PubMed and 61 unique publications in Web of Science were identified, and subsequently screened for triplet expansion, primary ovarian insufficiency, and/or early menopause. Of these, 56 papers were selected, excluding publications exclusively on males as well as studies on non-human species, pediatric cases, reviews, and expression studies. The study and control groups repeat numbers, ethnicity, and conclusions in the article are listed. Main results and the role of chance Expansion in CGG trinucleotide repeat length happens at female meiosis and the risk for expansion increases with an increasing number of CGG repeats. There is an intermediate zone between 45 and 55 CGG repeats when the allele may be stable or unstable. Among 56 publications, 54 describing FMR1 triplet repeat size in women with POI, 34 were studying only the women with POI (observational) while 20 were including also control group (comparative). 2 publications were meta-analyses in which 13 and 11 case control studies were included respectively, 5 studies being in common. POI-associated premutations showed a wide range of repeat sizes. Women carrying midsize range repeats (>70-<100) potentially have a higher risk for POI compared to the general population. In recent decades, population-based screenings have indicated that FMR1 premutations are not as prevalent in women with ovarian insufficiency as previous estimates have suggested, but they still represent a substantial cause of POI. When present, the number of AGG interruptions and the size of uninterrupted CGG repeats are directly correlated with the ovarian reserve. No increased risk of POI associated with a premutation was reported among populations of non-European descent, such as the Han Chinese, Indian, and Jordan populations. Limitations, reasons for caution Available reports are difficult to compare because the ethnicity and number of patients analyzed, availability of clinical data and the quality of results are different in each study. Additionally, an effect of X-inactivation in POI women with premutation was not studied. Wider implications of the findings Owing to the low penetrance and a molecular mechanism that has not yet been fully elucidated, the clinical utility of FMR1 screening in women with reduced ovarian reserve needs further investigation before clinical implication. Trial registration number N/A

Clinical implications of somatic allele expansion in female FMR1 premutation carriers

Carriers of a premutation allele (PM) in the FMR1 gene are at risk of developing a number of Fragile X premutation asssociated disorders (FXPAC), including Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), Fragile X-associated Primary Ovarian Insufficiency (FXPOI), and Fragile X-associated neuropsychiatric disorders (FXAND). We have recently reported somatic CGG allele expansion in female PM; however, its clinical significance remains unclear. The aim of this study was to examine the potential clinical association between somatic FMR1 allele instability and PM associated disorders. Participants comprised of 424 female PM carriers age 0.3– 90 years. FMR1 molecular measures and clinical information on the presence of medical conditions, were determined for all subjects for primary analysis. Two sub-groups of participants (age ≥ 25, N = 377 and age ≥ 50, N = 134) were used in the analysis related to presence of FXPOI and FXTAS, respectively. Among all participants (N = 424), the degree of instability (expansion) was significantly higher (median 2.5 vs 2.0, P = 0.026) in participants with a diagnosis of attention deficit hyperactivity disorder (ADHD) compared to those without. FMR1 mRNA expression was significantly higher in subjects with any psychiatric disorder diagnosis (P = 0.0017); specifically, in those with ADHD (P = 0.009), and with depression (P = 0.025). Somatic FMR1 expansion was associated with the presence of ADHD in female PM and FMR1 mRNA levels were associated with the presence of mental health disorders. The findings of our research are innovative as they suggest a potential role of the CGG expansion in the clinical phenotype of PM and may potentially guide clinical prognosis and management.

FMR1 alleles in women with idiopathic infertility

Introduction: The frequency of the premutated alleles of the FMR1 gene varies from 1: 100 to 1: 260 Israeli, Canadian, Finnish and American women, but it is unknown in Brazil. Premutation carriers may have reduced reproductive age and are at risk of transmitting the expanded allele to their offspring, and consequently fragile X syndrome. Objective: To observe the distribution range of the FMR1 gene alleles in a population of women with idiopathic infertility, without symptoms of premature ovarian insufficiency. Methods: The presence of premutation in FMR1 was assessed by conventional PCR, agarose and acrylamide gel and analysis of fragments in capillary electrophoresis. From the lymphocyte DNA obtained from 283 women undergoing infertility treatment. Results: It was observed that 169 patients had the normal heterozygous allele (59.7%), 114 had the normal homozygous allele (40.6%) and no patient had the premutation. Premature ovarian insufficiency is seen in 20 to 30% of women with the premutated allele. Thus, the condition can be asymptomatic in a large part of the premutation carriers. Brazil has a diverse population and, therefore, the allele frequencies of many gene variants are unknown. Previous Brazilian studies have shown a low frequency of the premutated allele in different patient cohorts. Corroborating these articles, the results demonstrated that the frequency of the premutated allele is low in the infertile women population studied. Conclusion: Tracking the size of the FMR1 gene alleles allows the expansion of knowledge about the frequency of risk alleles associated to genetic diseases in the Brazilian population.

Maternal FMR1 alleles expansion in newborns during transmission: a prospective cohort study.

The CGG repeats in the 5' untranslated region of the fragile X mental retardation 1 gene (FMR1) gene shows increased instability upon maternal transmission. Maternal FMR1 intermediate (45-54 repeats) and premutation (PM: 55-<200 repeats) alleles usually expand to full mutation (>200 repeats) alleles in offspring and consequently, cause fragile X syndrome (FXS) in them. In a prospective cohort study, Pakistani pregnant women in prenatal care were first screened for FMR1 expanded alleles. In the follow-up, pregnancy outcomes in women carrying FMR1 expanded alleles were recorded and their newborn offspring were also screened for FXS. In a total of 1950 pregnant women, 89 (4.6%) were detected carriers for FMR1 expanded alleles; however, rates of detection of expanded alleles were found significantly high in women with a history of FXS. In addition, miscarriages and birth of affected newborns with FXS were significantly more common in women carrying large size PM alleles and had a history of FXS (P = 0.0494 and P = 0.0494, respectively). The current study provides the first evidence of screening Pakistani pregnant women for FMR1 expanded alleles in prenatal care. Moreover, the miscarriage was also detected as a clinical predictor for FXS. Offspring would have a higher risk of developing FXS due to maternal FMR1 alleles expansions during transmission. This is the first prospective cohort study in Pakistan for finding FMR1 allelic status of pregnant women and their newborn offspring in follow-up. The robust offspring risk for FXS estimated in this study may be valuable information for genetic counseling of women carriers for FMR1 expanded alleles. The family history and miscarriage were detected as effective indicators for FXS carrier screening in Pakistani women.

Mechanisms of the FMR1 Repeat Instability: How Does the CGG Sequence Expand?

A dynamic mutation in exon 1 of the FMR1 gene causes Fragile X-related Disorders (FXDs), due to the expansion of an unstable CGG repeat sequence. Based on the CGG sequence size, two types of FMR1 alleles are possible: “premutation” (PM, with 56-200 CGGs) and “full mutation” (FM, with >200 triplets). Premutated females are at risk of transmitting a FM allele that, when methylated, epigenetically silences FMR1 and causes Fragile X syndrome (FXS), a very common form of inherited intellectual disability (ID). Expansions events of the CGG sequence are predominant over contractions and are responsible for meiotic and mitotic instability. The CGG repeat usually includes one or more AGG interspersed triplets that influence allele stability and the risk of transmitting FM to children through maternal meiosis. A unique mechanism responsible for repeat instability has not been identified, but several processes are under investigations using cellular and animal models. The formation of unusual secondary DNA structures at the expanded repeats are likely to occur and contribute to the CGG expansion. This review will focus on the current knowledge about CGG repeat instability addressing the CGG sequence expands.

Intermediate and expanded FMR1 alleles in an autistic Costa Rican population

Intermediate and expanded FMR1 alleles in an autistic Costa Rican population

High normal sized CGG repeat on the FMR1 gene reduces live birth rates after in vitro fertilization in Han Chinese

Substantial evidence now suggests an association between the FMR1 genotype and female fertility. The aim of this study was to determine whether a high normal FMR1 allele (35–54 repeats) affects in vitro fertilization (IVF) outcomes in Chinese women. A total of 120 women with 210 IVF cycles were retrospectively recruited in this study. The patients were divided into two groups based on the FMR1 repeat lengths at allele 2 (normal repeat group: <35 repeats; high repeat group: 35–54 repeats). The observed primary outcomes were the clinical pregnancy rate and live birth rate. No associations were observed between the high normal FMR1 allele and lower clinical pregnancy rate or live birth rate after adjusting for maternal age, education, work status, duration of infertility and number of embryos transferred (aOR 0.633, 95% CI 0.249–1.601, p = 0.337; aOR 0.325, 95% CI 0.094–1.118, p = 0.075; respectively). However, after additionally adjusting for anti-Müllerian hormone (AMH) level, there was a weak but significant association between high normal sized CGG repeats and a lower live birth rate (aOR 0.218, 95% CI 0.057–0.836, p = 0.026). The rate of available embryos showed a decreasing trend in patients with a high normal FMR1 allele, although the difference was not statistically significant after adjusting for maternal age, education, work status, duration of infertility and AMH level (aOR 0.905, 95% CI 0.810–1.011, p = 0.078). Furthermore, the number of CGG repeats in either allele was not associated with the live birth rate after adjusting for all confounding factors (aOR 0.832, 95% CI 0.677–1.023, p = 0.081; aOR 0.865, 95% CI 0.651–1.148, p = 0.315; respectively). In addition, no significant differences were found in the rates of good-quality embryos (p = 0.263), miscarriage (p = 0.861) or cycle cancellation (p = 0.295) between the groups. Taken together, in the Chinese population, individuals with high normal sized CGG repeats on the FMR1 gene have a higher risk of reduced live birth rates in childbearing age. Therefore, we recommend enhanced screening for fragile X syndrome in women of childbearing age in China. This study also suggests that the association between the FMR1 genotype and fertility in Chinese women merits further research.

High-Throughput Methylation-Specific Triplet-Primed PCR and Melting Curve Analysis for Selective and Reliable Identification of Actionable FMR1 Genotypes

Methylated FMR1 full-mutation expansions cause fragile X syndrome. FMR1 premutation carriers are susceptible to other late-onset conditions, and women with premutation are at risk of transmitting a fully expanded FMR1 allele to offspring. Identification of individuals with actionable FMR1 genotypes (full-mutation males and females, and premutation females at risk for primary ovarian insufficiency and/or having fragile X-affected offspring) can enable timely access to intervention services and genetic counseling. This study presents a rapid, first-tier test based on melting curve analysis of methylation-specific triplet-primed PCR amplicons (msTP-PCR MCA) for concurrent detection of FMR1 CGG-repeat expansions and their methylation status. The msTP-PCR MCA assay was optimized on 20 fragile X reference samples, and its performance was evaluated on 111 peripheral blood-derived DNA samples from patients who have undergone prior molecular testing with PCR and/or Southern blot analysis. The msTP-PCR MCA assay detected all samples with a methylated FMR1 CGG-repeat expansion, and had sensitivity, specificity, positive predictive value, and negative predictive values of 100%, 92.06%, 91.1%, and 100%, respectively. The msTP-PCR MCA assay identified premutation/full-mutation mosaicism down to 1%, detected skewed inactivation in females with FMR1 expansions, and enabled selective identification of all individuals with an actionable FMR1 genotype. The msTP-PCR MCA assay may aid in fragile X screening of at-risk populations and newborns and voluntary carrier screening of women of reproductive age.

The association between mosaicism type and cognitive and behavioral functioning among males with fragile X syndrome.

Mosaicism in fragile X syndrome (FXS) refers to two different FMR1 allele variations: size mosaicism represents different numbers of CGG repeats between the two alleles, such that in addition to a full mutation allele there is an allele in the normal or premutation range of CGG repeats, while methylation mosaicism indicates whether a full-mutation allele is fully or partially methylated. The present study explored the association between mosaicism type and cognitive and behavioral functioning in a large sample of males 3 years and older (n=487) with FXS, participating in the Fragile X Online Registry with Accessible Research Database. Participants with methylation mosaicism were less severely cognitively affected as indicated by a less severe intellectual disability rating, higher intelligence quotient and adaptive behavior score, and lower social impairment score. In contrast, the presence of size mosaicism was not significantly associated with better cognitive and behavioral outcomes than full mutation. Our findings suggest that methylation mosaicism is associated with better cognitive functioning and adaptive behavior and less social impairment. Further research could assess to what extent these cognitive and behavioral differences depend on molecular diagnostic methods and the impact of mosaicism on prognosis of individuals with FXS.

FMR1 allele frequencies in 51,000 newborns: a large-scale population study in China.

Fragile X syndrome (FXS), caused by CGG-repeat expansion in FMR1 promoter, is one of the most common causes of mental retardation. Individuals with full mutation and premutation alleles have a high risk of psychophysiological disorder and of having affected offspring. Frequencies of FMR1 alleles in general newborns have been reported in Caucasians but have not been investigated in the large-scale population in themainland ofChina. The sizes of FMR1 CGG-repeats were analyzed in 51,661 newborns (28,114 males and 23,547 females) and also in a cohort of 33 children diagnosed with developmental delay using GC-rich polymerase chain reaction (PCR) and triple repeat primed PCR. The frequency of CGG repeats > 100 was 1/9371 in males and 1/5887 in females, and the frequency of CGG repeats > 54 was 1/1561 in males and 1/1624 in females. FMR1 full mutation and premutation were identified in 27.27% of children who had Ages and Stages Questionnaire scores less than two standard deviations from the cutoff value. Our study revealed the prevalence of FXS in China and improved the sample databases of FXS, suggesting that the prevalence of FXS in Chinese is higher than estimated previously and that FXS screening can be advised to high-risk families.

Mechanisms of Genome Instability in the Fragile X-Related Disorders.

The Fragile X-related disorders (FXDs), which include the intellectual disability fragile X syndrome (FXS), are disorders caused by expansion of a CGG-repeat tract in the 5′ UTR of the X-linked FMR1 gene. These disorders are named for FRAXA, the folate-sensitive fragile site that localizes with the CGG-repeat in individuals with FXS. Two pathological FMR1 allele size classes are distinguished. Premutation (PM) alleles have 54–200 repeats and confer the risk of fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI). PM alleles are prone to both somatic and germline expansion, with female PM carriers being at risk of having a child with >200+ repeats. Inheritance of such full mutation (FM) alleles causes FXS. Contractions of PM and FM alleles can also occur. As a result, many carriers are mosaic for different sized alleles, with the clinical presentation depending on the proportions of these alleles in affected tissues. Furthermore, it has become apparent that the chromosomal fragility of FXS individuals reflects an underlying problem that can lead to chromosomal numerical and structural abnormalities. Thus, large numbers of CGG-repeats in the FMR1 gene predisposes individuals to multiple forms of genome instability. This review will discuss our current understanding of these processes.

OBSERVED OUTCOMES OF FMR1 PGT-M ANALYSIS WITH INCORPORATION OF CGG REPEAT EXPANSION CAN LEAD TO EXTRA EMBRYOS SUITABLE FOR TRANSFER

Highlight utility of triplet repeat expansion for the identification of embryos with the abnormal FMR1 allele that may be considered for transfer following preimplantation genetic testing (PGT) FMR1-related disorders, which includes Fragile X syndrome. Trophectoderm biopsies from US-based IVF clinics were sent to CooperGenomics for PGT-M for FMR1-related disorders between January 2018 and February 2021. Samples underwent linkage analysis via Karyomapping (lllumina, USA) and triplet repeat size determination. When requested, screening for aneuploidy (PGT-A) via next generation sequencing (NGS) was completed in conjunction with PGT-M. Patients with FMR1 expansions on both alleles and those with a full expansion (>200 repeats) on one allele were removed from the study population. A total of 1130 blastocysts from 318 IVF cycles underwent triplet repeat size determination and linkage analysis (average 3.55 embryos per cycle). The average age of patient at time of egg retrieval was 34.5 years. 51.4% (581/1130) of embryos were suitable to transfer based on presence of the normal FMR1 allele via linkage analysis. When factoring in PGT-A, 27.7% of the study population were euploid embryos with the normal allele. 48.6% (549/1130) of embryos tested had the abnormal FMR1 allele detected via linkage analysis. Of embryos inheriting the abnormal allele, 30.6% of embryos had experienced an expansion to yield an abnormal allele with >200 repeats (168/549). This expansion places these embryos at increased risk of expressing a FMR1-related disorder. Of these higher risk embryos, 55.4% were male. Importantly, 381 blastocyst samples (69.4%) inheriting the abnormal allele did not experience clinically significant expansion, with repeats remaining <200. 53% (202) of these lower risk embryos further screened as euploid by PGT-A, yielding an additional 17.9% (202/1130) embryos that could be considered for transfer. 49.25% (99/201) were female euploid carriers and 50.74% were male euploid carriers (102/201). Preimplantation testing for monogenic diseases (PGT-M) is classically considered a binary linkage-based test for the determination of the allele present in an at-risk embryo. For triplet repeat conditions like FMR1-related disorders, quantifying the length of the CGG triplet repeat tract inside the FMR1 gene modifies classification of disease risk in the embryo. This results in more embryos available for transfer, which is an important factor to consider in a population where ovarian failure is a common factor associated with carrier status. Knowledge of embryo repeat size is an intangible resource for genetic counseling about which embryos can be considered for transfer. Providing linkage + triplet repeat analysis is a diagnostic advantage for more complete classification of embryo status.

Diagnostic profile of the AmplideX Fragile X Dx and Carrier Screen Kit for diagnosis and screening of fragile X syndrome and other FMR1-related disorders

ABSTRACT Introduction: In 2009, a novel, CGG repeat primed FMR1 PCR assay was designed with primers flanking the triplet repeat region, as well as a third chimeric primer complementary to the (CGG)n repeat, that was capable of amplifying alleles throughout the repeat range. This assay for the first time allowed consistent detection of large full mutation alleles with PCR, resolution of heterozygosity in females and mapping of AGG interspersions. Areas Covered: The AmplideX Fragile X Dx and Carrier Screen Kit (Asuragen, Inc.) represents a refined assay that underwent validation with sensitivity analyses for FDA approval. Single-site precision, analytical sensitivity and specificity, limit of detection and diagnostic performance were assessed in comparison to reference methods at three independent sites. Single-site precision across all genotype categories showed 100% agreement at 20 ng input across multiple operators, days, instruments and kit lots. Compared to Southern Blot analysis, the overall percent agreement was over 98% for all expanded alleles. Expert Opinion: Limitations include no methylation assessment and hard to see full mutation peaks in some mosaic samples, but overall the assay is considered a highly accurate and time-efficient assay for FMR1 allele size determination.

Differential Progression of Motor Dysfunction Between Male and Female Fragile X Premutation Carriers Reveals Novel Aspects of Sex-Specific Neural Involvement.

Expansions of the CGG repeat in the non-coding segment of the FMR1 X-linked gene are associated with a variety of phenotypic changes. Large expansions (>200 repeats), which cause a severe neurodevelopmental disorder, the fragile x syndrome (FXS), are transmitted from the mothers carrying smaller, unstable expansions ranging from 55 to 200 repeats, termed the fragile X premutation. Female carriers of this premutation may themselves experience a wide range of clinical problems throughout their lifespan, the most severe being the late onset neurodegenerative condition called “Fragile X-Associated Tremor Ataxia Syndrome” (FXTAS), occurring between 8 and 16% of these carriers. Male premutation carriers, although they do not transmit expanded alleles to their daughters, have a much higher risk (40–50%) of developing FXTAS. Although this disorder is more prevalent and severe in male than female carriers, specific sex differences in clinical manifestations and progress of the FXTAS spectrum have been poorly documented. Here we compare the pattern and rate of progression (per year) in three motor scales including tremor/ataxia (ICARS), tremor (Clinical Tremor Rating scale, CRST), and parkinsonism (UPDRS), and in several cognitive and psychiatric tests scores, between 13 female and 9 male carriers initially having at least one of the motor scores ≥10. Moreover, we document the differences in each of the clinical and cognitive measures between the cross-sectional samples of 21 female and 24 male premutation carriers of comparable ages with FXTAS spectrum disorder (FSD), that is, who manifest one or more features of FXTAS. The results of progression assessment showed that it was more than twice the rate in male than in female carriers for the ICARS-both gait ataxia and kinetic tremor domains and twice as high in males on the CRST scale. In contrast, sex difference was negligible for the rate of progress in UPDRS, and all the cognitive measures. The overall psychiatric pathology score (SCL-90), as well as Anxiety and Obsessive/Compulsive domain scores, showed a significant increase only in the female sample. The pattern of sex differences for progression in motor scores was consistent with the results of comparison between larger, cross-sectional samples of male and female carriers affected with the FSD. These results were in concert with sex-specific distribution of MRI T2 white matter hyperintensities: all males, but no females, showed the middle cerebellar peduncle white matter hyperintensities (MCP sign), although the distribution and severity of these hyperintensities in the other brain regions were not dissimilar between the two sexes. In conclusion, the magnitude and specific pattern of sex differences in manifestations and progression of clinically recorded changes in motor performance and MRI lesion distribution support, on clinical grounds, the possibility of certain sex-limited factor(s) which, beyond the predictable effect of the second, normal FMR1 alleles in female premutation carriers, may have neuroprotective effects, specifically concerning the cerebellar circuitry.

Development and Validation of a Mathematical Model to Predict the Complexity of FMR1 Allele Combinations.

The polymorphic trinucleotide repetitive region in the FMR1 gene 5′UTR contains AGG interspersions, particularly in normal-sized alleles (CGG < 45). In this range repetitive stretches are typically interrupted once or twice, although alleles without or with three or more AGG interspersions can also be observed. AGG interspersions together with the total length of the repetitive region confer stability and hinder expansion to pathogenic ranges: either premutation (55 < CGG < 200) or full mutation (CGG > 200). The AGG interspersions have long been identified as one of the most important features of FMR1 repeat stability, being particularly important to determine expansion risk estimates in female premutation carriers. We sought to compute the combined AGG interspersion numbers and patterns, aiming to define FMR1 repetitive tract complexity combinations. A mathematical model, the first to compute this cumulative effect, was developed and validated using data from 131 young and healthy females. Plotting of their allelic complexity enabled the identification of two statistically distinct groups – equivalent and dissimilar allelic combinations. The outcome, a numerical parameter designated allelic score, depicts the repeat substructure of each allele, measuring the allelic complexity of the FMR1 gene including the AGGs burden, thus allowing new behavioral scrutiny of normal-sized alleles in females.

Effect of AGG Interruptions on FMR1 Maternal Transmissions.

There are four classes of CGG repeat alleles in the FMR1 gene: normal alleles have up to 44 repeats; patients with Fragile X Syndrome have more than 200 repeats; those between 55 and 200 CGGs are considered FMR1 premutation alleles, because they are associated with maternal expansions of the number of CGGs in the next generation and finally, alleles between 45 and 54 CGGs are called intermediate or gray zone alleles. In these last categories, the stability depends on the presence of AGG interruptions, which usually occurs between 9 and 10 CGGs. In this context, we have studied retrospectively 66 women with CGG repeats between 45 and 65, and their offspring. In total 87 transmissions were analyzed with triplet repeat primed PCR using AmplideX® FMR1 PCR (Asuragen, Austin, TX, USA) and we found that alleles with CGG repeats between 45 and 58 do not expand in the next generation except two cases with 56 repeats and 0 AGG interruptions. Furthermore, we have found four females with alleles with more than 59 CGG repeats and 2 AGG interruptions that do not expand either. Alleles from 56 CGG repeats without AGGs expand in all cases. In light of these results and those of the literature, we consider that the risk of unstable transmissions should be based on the presence or absence of AGG interruptions and not on the classical cutoffs which define each category of FMR1 alleles. The application of these results in the genetic and reproductive counseling is essential and AGG interruptions should always be studied.

Small Molecules Targeting H3K9 Methylation Prevent Silencing of Reactivated FMR1 Alleles in Fragile X Syndrome Patient Derived Cells.

In fragile X syndrome (FXS), expansion of a CGG repeat tract in the 5′-untranslated region of the FMR1 gene to >200 repeats causes transcriptional silencing by inducing heterochromatin formation. Understanding the mechanism of FMR1 silencing is important as gene reactivation is a potential treatment approach for FXS. To date, only the DNA demethylating drug 5-azadeoxycytidine (AZA) has proved effective at gene reactivation; however, this drug is toxic. The repressive H3K9 methylation mark is enriched on the FMR1 gene in FXS patient cells and is thus a potential druggable target. However, its contribution to the silencing process is unclear. Here, we studied the effect of small molecule inhibitors of H3K9 methylation on FMR1 expression in FXS patient cells. Chaetocin showed a small effect on FMR1 gene reactivation and a synergistic effect on FMR1 mRNA levels when used in combination with AZA. Additionally, chaetocin, BIX01294 and 3-Deazaneplanocin A (DZNep) were able to significantly delay the re-silencing of AZA-reactivated FMR1 alleles. These data are consistent with the idea that H3K9 methylation precedes DNA methylation and that removal of DNA methylation is necessary to see the optimal effect of histone methyl-transferase (HMT) inhibitors on FMR1 gene expression. Nonetheless, our data also show that drugs targeting repressive H3K9 methylation marks are able to produce sustained reactivation of the FMR1 gene after a single dose of AZA.

Incomplete silencing of full mutation alleles in males with fragile X syndrome is associated with autistic features

BackgroundFragile X syndrome (FXS) is a common monogenic cause of intellectual disability with autism features. While it is caused by loss of the FMR1 product (FMRP), mosaicism for active and inactive FMR1 alleles, including alleles termed premutation (PM: 55–199 CGGs), is not uncommon. Importantly, both PM and active full mutation (FM: ≥ 200 CGGs) alleles often express elevated levels of mRNA that are thought to be toxic. This study determined if complete FMR1 mRNA silencing from FM alleles and/or levels of FMR1 mRNA (if present) in blood are associated with intellectual functioning and autism features in FXS.MethodsThe study cohort included 98 participants (70.4% male) with FXS (FM-only and PM/FM mosaic) aged 1–43 years. A control group of 14 females were used to establish control FMR1 mRNA reference range. Intellectual functioning and autism features were assessed using the Mullen Scales of Early Learning or an age-appropriate Wechsler Scale and the Autism Diagnostic Observation Schedule-2nd Edition (ADOS-2), respectively. FMR1 mRNA was analysed in venous blood collected at the time of assessments, using the real-time PCR relative standard curve method.ResultsFemales with FXS had significantly higher levels of FMR1 mRNA (p < 0.001) than males. FMR1 mRNA levels were positively associated with age (p < 0.001), but not with intellectual functioning and autistic features in females. FM-only males (aged < 19 years) expressing FM FMR1 mRNA had significantly higher ADOS calibrated severity scores compared to FM-only males with completely silenced FMR1 (p = 0.011). However, there were no significant differences between these subgroups on intellectual functioning. In contrast, decreased levels of FMR1 mRNA were associated with decreased intellectual functioning in FXS males (p = 0.029), but not autism features, when combined with the PM/FM mosaic group.ConclusionIncomplete silencing of toxic FM RNA may be associated with autistic features, but not intellectual functioning in FXS males. While decreased levels of mRNA may be more predictive of intellectual functioning than autism features. If confirmed in future studies, these findings may have implications for patient stratification, outcome measure development, and design of clinical and pre-clinical trials in FXS.

Repeat Instability in the Fragile X-Related Disorders: Lessons from a Mouse Model.

The fragile X-related disorders (FXDs) are a group of clinical conditions that result primarily from an unusual mutation, the expansion of a CGG-repeat tract in exon 1 of the FMR1 gene. Mouse models are proving useful for understanding many aspects of disease pathology in these disorders. There is also reason to think that such models may be useful for understanding the molecular basis of the unusual mutation responsible for these disorders. This review will discuss what has been learnt to date about mechanisms of repeat instability from a knock-in FXD mouse model and what the implications of these findings may be for humans carrying expansion-prone FMR1 alleles.