Premutation Research Articles

Apolipoproteine and KLOTHO Gene Variants Do Not Affect the Penetrance of Fragile X-Associated Tremor/Ataxia Syndrome.

In this study, the potential role and interaction of the APOε and KLOTHO genes on the penetrance of fragile X-associated tremor/ataxia syndrome (FXTAS) and on the IQ trajectory were investigated. FXTAS was diagnosed based on molecular, clinical and radiological criteria. Males with the premutation (PM) over 50 years, 165 with and 34 without an FXTAS diagnosis, were included in this study and were compared based on their APO (ε2-ε3-ε4) and KLOTHO variant (KL-VS) genotypes. The effect of APOε4 on FXTAS stage and on diagnosis did not differ significantly by KL-VS genotype with interaction effect p = 0.662 and p = 0.91, respectively. In the FXTAS individuals with an APOε2 allele, a marginal significance was observed towards a larger decline in verbal IQ (VIQ) in individuals with an APOε4 allele compared to those without an APOε4 allele (p = 0.071). In conclusion, our findings suggest that the APOε4 and KL-VS genotypes alone or through their interaction effect do not appear to predispose to either FXTAS diagnosis or stage in male carriers of the PM allele. A further study is needed to establish the trend of IQ decline in the FXTAS individuals who carry APOε4 with APOε2 compared to those without APOε4.

Intrasubject variability of sustained attention is associated with elevated self-reported attention deficits in women with a fragile X premutation allele.

Women with a fragile X premutation (PM) self-report higher rates of attention difficulties than women without a PM; however, results of studies using objective measures of attention are inconsistent. The present study assessed whether intrasubject variability during a sustained attention task better predicted functional outcomes in women with a PM than the previously published standard reaction time and accuracy variables. We analyzed continuous performance test, a computerized measure of sustained attention, and the Conners' Adult Attention-Deficit/Hyperactivity Disorder Rating Scale Report (CAARS) data from 273 women with a PM and 175 women without a PM aged 18-50 years. Separate analyses using Pearson correlations and independent t tests were performed on the full range of coefficient of variation (CV) of reaction time scores and the subset of scores that showed higher variability. Performance variability of sustained attention measured by the continuous performance test was associated with functional outcomes measured by the CAARS in women with a PM but not women without a PM. Specifically, the CV in those with higher variability was correlated with two CAARS subscale scores (p = .006). Independent t tests showed significant differences in CV between CAARS scores dichotomized for the presence of subclinical symptoms for two subscales (p ≤ .001-.007). Correlation between the full range of CV scores and the CAARS Inattention/Memory Problems subscale approached significance (p = .012). Findings highlight the importance of including intrasubject variability in analyzing attention in clinical populations as a more sensitive objective measure associated with reported symptoms and to assist in predicting functional outcomes. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Ovarian reserve in patients with FMR1 gene premutation and the role of fertility preservation

IntroductionWomen with premutation (PM) of the FMR1 gene may suffer from reduced ovarian reserve or even premature ovarian insufficiency (POI). We studied hormonal and ultrasound ovarian reserve, fertility and fertility preservation outcomes in these patients. Patients and methodRetrospective cohort study of 63 female FMR1 premutation carriers. ResultsSixty-three female patients bearing an FMR1 premutation were included. Median age was 30 years [26.5–35]. Median number of CGG triplets was 83 [77.2–92]. Before diagnosis of PM, 19 women (30%) had had in all 35 pregnancies, resulting in 20 births, including 7 affected children. After diagnosis of PM, 17 women (26.1%) had in all 23 pregnancies, at a median age of 34.5 years [32.2–36.0]: 2 after pre-implantation genetic diagnosis, 3 after oocyte donation, 18 spontaneously, and 5 ending in medical termination for fragile X syndrome. Thirty-three patients (52.4%) had POI diagnosis (median age, 30 years [27–34]) with median FSH level 84 IU/L [50.5–110] and median AMH level 0.08ng/mL [0.01–0.19]. After POI diagnosis, 8 women had in all 9 pregnancies: 3 following oocyte donation, and 6 spontaneous in 5 women (15.1%). Eight of the 9 pregnancies resulted in a live birth (including 2 affected children) and 1 in medical termination for trisomy 13. The median age of the 30 patients without POI was 31 years [25.2–35.0].Thirteen women (20.6%) underwent fertility preservation, at a median age of 29 years [24–33]: FSH 7.7 IU/L [6.8–9.9], AMH 1.1ng/mL [0.95–2.1], antral follicle count 9.5 [7.7–14.7]. A median 15 oocytes [10–26] were cryopreserved in a median 2 cycles [1–3]. At the time of writing, no oocytes had yet been thawed for in-vitro fertilization. ConclusionsThis study shows the importance of early fertility preservation after diagnosis of FMR1 premutation in women, due to early deterioration of ovarian reserve. Genetic counseling is essential in these patients, as spontaneous pregnancies are not uncommon, even in cases of impaired ovarian reserve, and can lead to birth of affected children.

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.

Healthcare Experiences of African American Women with the Fragile X Premutation.

This study aims to understand the healthcare experiences of African American women with a fragile X premutation (PM). PM carriers are at risk for fragile X-associated conditions, including primary ovarian insufficiency (FXPOI) and neuropsychiatric disorders (FXAND). There is no racial/ethnic association with carrying a PM, but African American women historically experience barriers receiving quality healthcare in the USA. Obstacles to care may increase mental health conditions like anxiety and depression. Eight African American women with a PM were interviewed to explore disparities in receiving healthcare and to learn about psychosocial experiences during and after their diagnoses. Interviews were transcribed verbatim and independently coded by two researchers. A deductive-inductive approach was used, followed by thematic analysis to determine prominent themes. The average participant age was 52.3 ± 8.60 years, with a mean age at premutation diagnosis of 31 ± 5.95 years. Seven participants had children with FXS. Themes from interviews included healthcare experiences, family dynamics, and emotional/mental health after their diagnosis. Participants reported concerns about not being taken seriously by providers and mistrust of the medical institutions. Within families, participants reported denial, insensitivity, and isolation. Participants reported a high incidence of anxiety and depression. Both are symptoms of FXAND and stresses of systemic racism and sexism. The reported family dynamics around the news of a genetic diagnosis stand apart from other racial cohorts in fragile X research: interventions like family counseling sessions and inclusive support opportunities from national organizations could ease the impacts of a PM for African American women.

Activation Ratio Correlates with IQ in Female Carriers of the FMR1 Premutation.

Carriers of the FMR1 premutation (PM) allele are at risk of one or more clinical conditions referred to as FX premutation-associated conditions (FXPAC). Since the FMR1 gene is on the X chromosome, the activation ratio (AR) may impact the risk, age of onset, progression, and severity of these conditions. The aim of this study was to evaluate the reliability of AR measured using different approaches and to investigate potential correlations with clinical outcomes. Molecular and clinical assessments were obtained for 30 PM female participants, and AR was assessed using both Southern blot analysis (AR-Sb) and methylation PCR (AR-mPCR). Higher ARs were associated with lower FMR1 transcript levels for any given repeat length. The higher AR-Sb was significantly associated with performance, verbal, and full-scale IQ scores, confirming previous reports. However, the AR-mPCR was not significantly associated (p > 0.05) with these measures. Similarly, the odds of depression and the number of medical conditions were correlated with higher AR-Sb but not correlated with a higher AR-mPCR. This study suggests that AR-Sb may be a more reliable measure of the AR in female carriers of PM alleles. However, further studies are warranted in a larger sample size to fully evaluate the methylation status in these participants and how it may affect the clinical phenotype.

Social and physical predictors of mental health impact in adult women who have an FMR1 premutation

PurposeClear understanding of mental health phenotypes and associated socioeconomic, physical health and well-being impacts in adult women with an FMR1 premutation (PM) is needed for counseling and primary healthcare. MethodsA questionnaire captured mental health conditions in women with a PM, using lifetime diagnosis and nested psychometric scales (Liebowitz Social Anxiety Scale; Depression and Anxiety Stress Scale). Socioeconomic, physical health and well-being data were entered into 2 multivariable logistic regression models (1 with depression status as the outcome variable and 1 with social anxiety as the outcome). Results137 participants were included. Depression was found in ∼30% and social anxiety in ∼38%. With depression status the outcome variable, strongest associations were for low education, diagnosis of migraine, diagnosis of irritable bowel syndrome, and self-reported hearing loss symptoms. With social anxiety status as the outcome, strongest associations were with education, migraine, irritable bowel syndrome, relationship status, and subjective memory complaints. ConclusionMental health impacts were found in more than 1 in 3 women. Poor mental health was significantly associated with socioeconomic and physical health factors. Findings may inform health care professionals about personalized treatment options for women with a PM.

Granulosa Cell Dysfunction Is Associated With Diminished Ovarian Response in FMR1 Premutation Carriers

FMR1 premutation (PM) carriers are at increased risk of ovarian impairment resulting in diminished ovarian response (DOR) to exogenous follicle-stimulating hormone (FSH) stimulation. Expanded CGG repeat transcript and RAN-associated protein (FMRpolyG) have been shown to accumulate in cellular aggregates and sequester proteins, thus impairing their function. Sam68 is a multifunctional RNA-binding protein highly expressed in the gonads involved in FSH receptor (FSHR) transcript maturation during FSH-dependent follicular development. The present study examined a possible pathophysiological explanation for DOR to exogenous FSH stimulation in FMR1 PM carriers. We used both a human granulosa cell (GC) line model and human GCs from FMR1 PM carriers to evaluate whether Sam68 is sequestered with expanded CGG repeat transcript. We show that Sam68 is sequestered in GCs, most likely by interaction with the expanded CGG repeat transcript. The sequestration may lead to reduced levels of free Sam68 available for FHSR precursor transcript processing, causing dysregulation of FSHR transcript maturation, and a consequent decrease in FSHR protein levels. Sam68 sequestration may underlie the diminished ovarian response to FSH stimulation in FMR1 PM carriers.

Non-canonical DNA/RNA structures associated with the pathogenesis of Fragile X-associated tremor/ataxia syndrome and Fragile X syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X syndrome (FXS) are primary examples of fragile X-related disorders (FXDs) caused by abnormal expansion of CGG repeats above a certain threshold in the 5′-untranslated region of the fragile X mental retardation (FMR1) gene. Both diseases have distinct clinical manifestations and molecular pathogenesis. FXTAS is a late-adult-onset neurodegenerative disorder caused by a premutation (PM) allele (CGG expansion of 55–200 repeats), resulting in FMR1 gene hyperexpression. On the other hand, FXS is a neurodevelopmental disorder that results from a full mutation (FM) allele (CGG expansions of ≥200 repeats) leading to heterochromatization and transcriptional silencing of the FMR1 gene. The main challenge is to determine how CGG repeat expansion affects the fundamentally distinct nature of FMR1 expression in FM and PM ranges. Abnormal CGG repeat expansions form a variety of non-canonical DNA and RNA structures that can disrupt various cellular processes and cause distinct effects in PM and FM alleles. Here, we review these structures and how they are related to underlying mutations and disease pathology in FXS and FXTAS. Finally, as new CGG expansions within the genome have been identified, it will be interesting to determine their implications in disease pathology and treatment.

P-516 Cell-type specific role of Fragile X Mental Retardation polyglycine protein in blood of women with Fragile X Mental Retardation 1 gene premutation

Abstract Study question What cell type is specific for the expression of FMRpolyG of FMR1 premutation (PM) in peripheral mononuclear blood cells (PMBC) and how can this impact female fertility? Summary answer The expression of FMRpolyG is significantly higher in T cells from peripheral blood mononuclear cells (PBMCs) of FMR1 PM carriers. What is known already Fragile X-associated primary ovarian insufficiency (FXPOI) is characterized by oligo/amenorrhea and hypergondotropic hypogonadism and is caused by expansion of the CGG-repeat in the 5'UTR of FMR1. Autoimmunity is highly associated with POI and in FMR1-PM activated inflammatory state and immune response have been discovered. RAN-translation dependent on variable CGG-repeat length is thought to cause FXPOI due to production of the polyglycine-containing-FMR1-protein, FMRpolyG. Colocalization of ubiquitin and FMRpolyG was recently demonstrated in ovarian stromal and mural granulosa cells from FMR1 PM carriers. Our previous data have also shown that FMRpolyG is aggregated in ubiquitin-positive inclusions in PBMCs from PM carriers. Study design, size, duration PBMCs and granulosa cells (GCs) from women with PM (5) and women without PM (10) (controls) were examined by immunofluorescence staining (IF) for the presence of inclusions positive for ubiquitin and FMRpolyG. Cell lysis and protein extraction samples were subjected to Western blot analysis (WB) to detect FMRP and FMRpolyG. Flow cytometric analyzes (FACs) were performed to determine cell type-specific expression of FMRpolyG. Participants/materials, setting, methods PBMCs were isolated from peripheral blood using Ficoll-Paque. PBMCs were fixed, IF stained for FMRpolyG and ubiquitin, and analyzed by fluorescence microscopy. WB was used to detect the expression of FMRP, FMRpolyG in extracted protein from lymphocytes and GCs. PBMCs were surface stained with CD3, CD14 and CD19 antibodies and later intracellular stained with FMRpolyG and ubiquitin for FACs analysis. Main results and the role of chance FMRpolyG aggregates were detected as ubiquitin-positive inclusions in PBMCs from PM carriers, whereas only weak signal without inclusions was detected in controls. We detected FMRpolyG as a 15- to 25-kDa protein in PBMCs from two FMR1 PM carriers with 124 and 81 CGG repeats, a PM range that is supposed to carry the highest risk to develop FXPOI within all PM carriers. Using FACs, we found that the expression levels of FMRpolyG were significantly higher in the FMR1 PM carriers than in the group without PM (3.5-fold, p = 0.03). Both FMR1 PM and non-PM groups showed normal distribution of T cells (58.38% ± 11.54 and 58.72% ± 6.16, respectively). However, FMRpolyG fluorescence intensity was 28.98-fold higher (p = 0.01) in the T cells of FMR1 PM carriers than in those of women without PM. FMRpolyG expression levels of B cells (CD19+) and monocytes (CD14+) were comparable in both groups. Of note, the ubiquitin-positive cells of FMR1 PM carriers had significantly higher expression levels of FMRpolyG than those of non-PM carriers (17.84-fold, p < 0.0001). Limitations, reasons for caution More patients are needed to support the present results. Further studies on the possible consequences of these FMRpolyG-positive inclusions in PM carriers are also advisable. Wider implications of the findings We found for the first time FMRpolyG aggregates in the peripheral blood of PM-carriers and a significant increase in FMRpolyG expression in T cells from PM-carriers. These results suggest that FMRpolyG may have a toxic potential and play an immunological role in ovarian damage, finally triggering the development of FXPOI. Trial registration number RE 3647/1-1 and /1-2

Both cis and trans-acting genetic factors drive somatic instability in female carriers of the FMR1 premutation

The fragile X mental retardation (FMR1) gene contains an expansion-prone CGG repeat within its 5′ UTR. Alleles with 55–200 repeats are known as premutation (PM) alleles and confer risk for one or more of the FMR1 premutation (PM) disorders that include Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), Fragile X-associated Primary Ovarian Insufficiency (FXPOI), and Fragile X-Associated Neuropsychiatric Disorders (FXAND). PM alleles expand on intergenerational transmission, with the children of PM mothers being at risk of inheriting alleles with > 200 CGG repeats (full mutation FM) alleles) and thus developing Fragile X Syndrome (FXS). PM alleles can be somatically unstable. This can lead to individuals being mosaic for multiple size alleles. Here, we describe a detailed evaluation of somatic mosaicism in a large cohort of female PM carriers and show that 94% display some evidence of somatic instability with the presence of a series of expanded alleles that differ from the next allele by a single repeat unit. Using two different metrics for instability that we have developed, we show that, as with intergenerational instability, there is a direct relationship between the extent of somatic expansion and the number of CGG repeats in the originally inherited allele and an inverse relationship with the number of AGG interruptions. Expansions are progressive as evidenced by a positive correlation with age and by examination of blood samples from the same individual taken at different time points. Our data also suggests the existence of other genetic or environmental factors that affect the extent of somatic expansion. Importantly, the analysis of candidate single nucleotide polymorphisms (SNPs) suggests that two DNA repair factors, FAN1 and MSH3, may be modifiers of somatic expansion risk in the PM population as observed in other repeat expansion disorders.

Fragile X premutation carrier screening in Pakistani preconception women in primary care consultation

PurposeWomen of reproductive age who carry fragile X premutation (PM) alleles have 56 to 200 CGG repeats in the 5′-untranslated region of FMR1 gene are at increased risk for producing children with intellectual disabilities (ID) or autism spectrum disorders (ASD) due to expansion of PM alleles to full mutation alleles (> 200 repeats) during maternal transmission.MethodsIn present study fragile X PM carrier screening was performed in total 808 women who were consulting primary health care centers for preconception care in Khyber Pakhtunkhwa region of Pakistan between April, 2018 and December, 2020. Polymerase chain reaction (PCR) was performed for detection of PM carrier women and the CGG repeats number was confirmed by Southern blotting and capillary electrophoresis.ResultsThe prevalence rate for PM carriers among preconception women was found to be 0.7% that was contributed by 0.5% women in risk group (RG1) with family history of ID and 0.2% in risk group 2 (RG2) with family history of ASD. PM carrier women had at least one affected child or sibling. In addition, the preconception women with FMR1 PM alleles were found to be at increased risk for primary ovary insufficiency (RG1: P = 0.0265, RG2: P = 0.0389), postpartum depression (RG1: P = 0.0240, RG2: P = 0.0501) and neuropsychiatric disorders (RG1: P = 0.0389, RG2: P = 0.0432).ConclusionsCurrent study provides first evidence of fragile X PM carrier screening in Pakistani preconception women in primary care consultation. Findings of current study may help to improve preconception care and to reduce burden of fragile X associated disorders in our population.

Expression of FMRpolyG in Peripheral Blood Mononuclear Cells of Women with Fragile X Mental Retardation 1 Gene Premutation

Fragile X-associated primary ovarian insufficiency (FXPOI) is characterized by oligo/amenorrhea and hypergonadotropic hypogonadism and is caused by the expansion of the CGG repeat in the 5′UTR of Fragile X Mental Retardation 1 (FMR1). Approximately 20% of women carrying an FMR1 premutation (PM) allele (55–200 CGG repeat) develop FXPOI. Repeat Associated Non-AUG (RAN)-translation dependent on the variable CGG-repeat length is thought to cause FXPOI, due to the production of a polyglycine-containing FMR1 protein, FMRpolyG. Peripheral blood monocyte cells (PBMCs) and granulosa cells (GCs) were collected to detect FMRpolyG and its cell type-specific expression in FMR1 PM carriers by immunofluorescence staining (IF), Western blotting (WB), and flow cytometric analysis (FACS). For the first time, FMRpolyG aggregates were detected as ubiquitin-positive inclusions in PBMCs from PM carriers, whereas only a weak signal without inclusions was detected in the controls. The expression pattern of FMRpolyG in GCs was comparable to that in the lymphocytes. We detected FMRpolyG as a 15- to 25-kDa protein in the PBMCs from two FMR1 PM carriers, with 124 and 81 CGG repeats. Flow cytometric analysis revealed that FMRpolyG was significantly higher in the T cells from PM carriers than in those from non-PM carriers. The detection of FMRpolyG aggregates in the peripheral blood and granulosa cells of PM carriers suggests that it may have a toxic potential and an immunological role in ovarian damage in the development of FXPOI.

Allopregnanolone Improves Locomotor Activity and Arousal in the Aged CGG Knock-in Mouse Model of Fragile X-Associated Tremor/Ataxia Syndrome.

Carriers of the fragile X premutation (PM) can develop a variety of early neurological symptoms, including depression, anxiety and cognitive impairment as well as being at risk for developing the late-onset fragile X-associated tremor/ataxia syndrome (FXTAS). The absence of effective treatments for FXTAS underscores the importance of developing efficacious therapies to reduce the neurological symptoms in elderly PM carriers and FXTAS patients. A recent preliminary study reported that weekly infusions of Allopregnanolone (Allop) may improve deficits in executive function, learning and memory in FXTAS patients. Based on this study we examined whether Allop would improve neurological function in the aged CGG knock-in (CGG KI) dutch mouse, B6.129P2(Cg)-Fmr1tm2Cgr/Cgr, that models much of the symptomatology in PM carriers and FXTAS patients. Wild type and CGG KI mice received 10 weekly injections of Allop (10 mg/kg, s.c.), followed by a battery of behavioral tests of motor function, anxiety, and repetitive behavior, and 5-bromo-2′-deoxyuridine (BrdU) labeling to examine adult neurogenesis. The results provided evidence that Allop in CGG KI mice normalized motor performance and reduced thigmotaxis in the open field, normalized repetitive digging behavior in the marble burying test, but did not appear to increase adult neurogenesis in the hippocampus. Considered together, these results support further examination of Allop as a therapeutic strategy in patients with FXTAS.

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.

Predictors of Comorbid Conditions in Women Who Carry an FMR1 Premutation.

Purpose: Women who carry an FMR1 premutation (PM) can experience two well-established PM-associated disorders: fragile X-associated primary ovarian insufficiency (FXPOI, affects ~20–30% carriers) and fragile X-associated tremor-ataxia syndrome (FXTAS, affects ~6–15% carriers); however, emerging evidence indicates that some of these women experience complex health profiles beyond FXPOI and FXTAS.Methods: In an effort to better understand predictors for these comorbid conditions, we collected self-reported medical histories on 413 women who carry an FMR1 PM.Results: There were 22 health conditions reported by at least 9% of women. In an exploratory analysis, 12 variables were tested in logistic regression models for each comorbid condition, including demographic variables, environmental variables, PM-associated factors, and endorsement of depression and/or anxiety. More than half of the comorbid conditions studied were associated with women who self-reported having anxiety. Age, smoking, body mass index (BMI), and depression were also significant predictor variables for specific comorbid conditions.Conclusions: Age, smoking, and BMI were significantly associated with a subset of the comorbid conditions analyzed. Importantly, depression or anxiety were also significantly associated with many of the comorbid health conditions. This work highlights some of the modifiable factors associated with complex health profiles among women with an FMR1 PM.

The Phenotypic Profile Associated With the FMR1 Premutation in Women: An Investigation of Clinical-Behavioral, Social-Cognitive, and Executive Abilities.

The FMR1 gene in its premutation (PM) state has been linked to a range of clinical and subclinical phenotypes among FMR1 PM carriers, including some subclinical traits associated with autism spectrum disorder (ASD). This study attempted to further characterize the phenotypic profile associated with the FMR1 PM by studying a battery of assessments examining clinical-behavioral traits, social-cognitive, and executive abilities in women carrying the FMR1 PM, and associations with FMR1-related variability. Participants included 152 female FMR1 PM carriers and 75 female controls who were similar in age and IQ, and screened for neuromotor impairments or signs of fragile X-associated tremor/ataxia syndrome. The phenotypic battery included assessments of ASD-related personality and language (i.e., pragmatic) traits, symptoms of anxiety and depression, four different social-cognitive tasks that tapped the ability to read internal states and emotions based on different cues (e.g., facial expressions, biological motion, and complex social scenes), and a measure of executive function. Results revealed a complex phenotypic profile among the PM carrier group, where subtle differences were observed in pragmatic language, executive function, and social-cognitive tasks that involved evaluating basic emotions and trustworthiness. The PM carrier group also showed elevated rates of ASD-related personality traits. In contrast, PM carriers performed similarly to controls on social-cognitive tasks that involved reliance on faces and biological motion. The PM group did not differ from controls on self-reported depression or anxiety symptoms. Using latent profile analysis, we observed three distinct subgroups of PM carriers who varied considerably in their performance across tasks. Among PM carriers, CGG repeat length was a significant predictor of pragmatic language violations. Results suggest a nuanced phenotypic profile characterized by subtle differences in select clinical-behavioral, social-cognitive, and executive abilities associated with the FMR1 PM in women.

P–514 RAN-Translation in Fragile X associated Premature Ovarian Insufficiency (FXPOI): FMRpolyG as a predictive tool

Abstract Study question Does repeat-associated non-AUG (RAN) translation lead to accumulation of polyglycine- containing protein (FMRpolyG) in human lymphocytes and mural granulosa cells of FMR1 premutation carriers? Summary answer Lymphocytes and granulosa cells from FMR1 premutation carriers contain intracellular inclusions that stain positive for both FMRpolyG and ubiquitin. What is known already: Fragile-X-associated-Primary-Ovarian-Insufficiency (FXPOI) is characterized by oligo/amenorrhea and hypergonadotropic hypogonadism associated with the expansion of CGG-repeats in the 5’UTR of FMR1, called premutation (PM) (n: 55–200). Approximately 20% of women carrying a FMR1-premutation (PM) allele develop FXPOI. RAN-translation dependent on variable CGG-repeat length is hypothesized to cause FXPOI due to the production of a polyglycine-containing FMR1-protein, FMRpolyG. Recently, FMRpolyG inclusions were found in neuronal brain cells of FXTAS patients and stromal cells of the ovary of an FXPOI patient. Study design, size, duration: Lymphocytes and granulosa cells (GCs) from women with PM (6) and women without PM (10) (controls) were analyzed by immunofluorescence (IF) staining for the presence of inclusions positive for ubiquitin and FMRpolyG. Cell lysis and protein extraction samples were subjected to Fluorescent Western Blot (WB) analysis to detect FMRP and FMRpolyG Participants/materials, setting, methods Human GCs were obtained from follicular fluid after oocyte retrieval and lymphocytes were isolated from peripheral blood using Ficoll-Paque. Cells suspended in PBS were adhered to a glass-coverslip placed at the bottom of the 6-well culture plate, via gravity sedimentation. Adhered cells were fixed, IF staining for FMRpolyG and ubiquitin was performed and analyzed by fluorescence microscopy. Fluorescent WB was used to demonstrate the expression of FMRP, FMRpolyG in extracted protein from lymphocytes and GCs. Main results and the role of chance FMRP was successfully detected by fluorescence WB in both lymphocytes and GCs. FMRP is mainly present in cytoplasm and was expressed in greater amount in GCs than in leukocytes. Moreover, FMRP expression was significantly decreased in GCs from FMR1-PM compared with controls. Lymphocytes from PM-carriers and controls were immunostained for FMRpolyG and ubiquitin. In PM-carriers, FMRpolyG was present as aggregates, whereas in controls only a weak signal without inclusions was detectable. The expression pattern of FMRpolyG in GCs was similar to that in lymphocytes with a significant increase in PM-carriers. There, the FMRpolyG-aggregates additionally demonstrated as ubiquitin-positive inclusions. These may resemble the toxic potential of these protein fractions involved the ovarian damage in developing FXPOI. Limitations, reasons for caution More patients are needed to support the present findings. Further investigation into the possible consequences of these FMRpolyG-positive inclusions in PM-carriers is also advisable. Wider implications of the findings: We found for the first time FMRpolyG-accumulation in lymphocytes and GCs from FMR1-PM-carriers in ubiquitin-positive inclusions. Future experiments evaluating consistency in more patients and elucidating the impact on fertility and prospective value for individual ovarian reserve are therefore in preparation. Trial registration number Not applicable

Accuracy and Performance Evaluation of Triplet Repeat Primed PCR as an Alternative to Conventional Diagnostic Methods for Fragile X Syndrome

BackgroundConventional diagnosis of fragile X syndrome (FXS) is based on a combination of fragment analysis (FA) and Southern blotting (SB); however, this diagnostic approach is time- and labor-intensive and has pitfalls such as the possibility of missing large number alleles. Triplet repeat primed PCR (TP-PCR) is a current alternative used to overcome these limitations. We evaluated the diagnostic usefulness of TP-PCR compared with the conventional diagnostic protocol consisting of FA and/or SB in terms of allele categorization, repeat number correlation, and zygosity concordance in female genetic carriers.MethodsFrom November 2013 to March 2018, 458 patients (326 males, 132 females) were simultaneously examined using FA and/or SB and TP-PCR by detecting CGG repeat numbers in FMR1 gene and diagnosed as per American College of Medical Genetics guidelines.ResultsThe TP-PCR results showed high concordance with the FA and/or SB results for all three aspects (allele categorization, repeat number correlation, and zygosity concordance in female genetic carriers). TP-PCR detected CGG expansions ≥200 in all full mutation (FM) allele cases in male patients, as well as both the normal allele (NL) and FM allele in female carriers. In premutation (PM) allele carriers, the TP-PCR results were consistent with the FA and/or SB results. In terms of zygosity concordance in female genetic carriers, 12 NL cases detected by TP-PCR showed a merged peak consisting of two close heterozygous peaks; however, this issue was resolved using a 10-fold dilution.ConclusionsTP-PCR may serve as a reliable alternative method for FXS diagnosis.

Identifying susceptibility genes for primary ovarian insufficiency on the high-risk genetic background of a fragile X premutation

Objective:To identify modifying genes that explains the risk of fragile X-associated primary ovarian insufficiency (FXPOI).Design:Gene-based, case/control association study, followed by a functional screen of highly ranked genes using a Drosophila model.Setting:Participants were recruited from academic and clinical settings.Patient(s):Women with a premutation (PM) who experienced FXPOI at the age of 35 years or younger (n = 63) and women with a PM who experienced menopause at the age of 50 years or older (n = 51) provided clinical information and a deoxyribonucleic acid sample for whole genome sequencing. The functional screen was on the basis of Drosophila TRiP lines.Intervention(s):Clinical information and a DNA sample were collected for whole genome sequencing.Main Outcome Measures:A polygenic risk score derived from common variants associated with natural age at menopause was calculated and associated with the risk of FXPOI. Genes associated with the risk of FXPOI were identified on the basis of the P-value from gene-based association test and an altered level of fecundity when knocked down in the Drosophila PM model.Results:The polygenic risk score on the basis of common variants associated with natural age at menopause explained approximately 8% of the variance in the risk of FXPOI. Further, SUMO1 and KRR1 were identified as possible modifying genes associated with the risk of FXPOI on the basis of an untargeted gene analysis of rare variants.Conclusions:In addition to the large genetic effect of a PM on ovarian function, the additive effects of common variants associated with natural age at menopause and the effect of rare modifying variants appear to play a role in FXPOI risk.