Blythe Durbin-Johnson

AGG interruptions within the maternal FMR1 gene reduce the risk of offspring with fragile X syndrome.

Purpose:The ability to accurately predict the likelihood of expansion of the CGG repeats in the FMR1 gene to a full mutation is of critical importance for genetic counseling of women who are carriers of premutation alleles (55–200 CGG repeats) and who are weighing the risk of having a child with fragile X syndrome. The presence of AGG interruptions within the CGG repeat tract is thought to decrease the likelihood of expansion to a full mutation during transmission, thereby reducing risk, although their contribution has not been quantified.Methods:We retrospectively analyzed 267 premutation alleles for number and position of AGG interruptions, length of pure CGG repeats, and CGG repeat lengths present in the offspring of the maternal transmissions. In addition, we determined the haplotypes of four markers flanking the 5′-UTR locus in the premutation mothers.Results:We found that the presence of AGG interruptions significantly increased genetic stability, whereas specific haplotypes had a marginal association with transmission instability.Conclusion:The presence of AGG interruptions reduced the risk of transmission of a full mutation for all maternal (premutation) repeat lengths below ~100 CGG repeats, with a differential risk (0 vs. 2 AGG) exceeding 60% for alleles in the 70- to 80-CGG repeat range.Genet Med 2012:14(8):729–736

High MMP-9 activity levels in fragile X syndrome are lowered by minocycline

Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by lack of the FMR1 protein, FMRP, a translational repressor. Its absence leads to up‐regulation of locally translated proteins involved in synaptic transmission and plasticity, including the matrix metalloproteinase‐9 (MMP‐9). In the Fmr1 knock‐out (KO), a mouse model of FXS, an abnormal elevated expression of MMP‐9 in the brain was pharmacologically down‐regulated after treatment with the tetracycline derivative minocycline. Moreover, the rescue of immature dendritic spine morphology and a significant improvement of abnormal behavior were associated with down‐regulation of MMP‐9. Here, we report on high plasma activity of MMP‐9 in individuals with FXS. In addition, we investigate MMP‐9 changes in patients with FXS who have gone through a minocycline controlled clinical trial and correlate MMP‐9 activity to clinical observations. The results of this study suggest that, in humans, activity levels of MMP‐9 are lowered by minocycline and that, in some cases, changes in MMP‐9 activity are positively associated with improvement based on clinical measures.

Transcriptomic Analysis of Toxoplasma Development Reveals Many Novel Functions and Structures Specific to Sporozoites and Oocysts

Sexual reproduction of Toxoplasma gondii occurs exclusively within enterocytes of the definitive felid host. The resulting immature oocysts are excreted into the environment during defecation, where in the days following, they undergo a complex developmental process. Within each oocyst, this culminates in the generation of two sporocysts, each containing 4 sporozoites. A single felid host is capable of shedding millions of oocysts, which can survive for years in the environment, are resistant to most methods of microbial inactivation during water-treatment and are capable of producing infection in warm-blooded hosts at doses as low as 1–10 ingested oocysts. Despite its extremely interesting developmental biology and crucial role in initiating an infection, almost nothing is known about the oocyst stage beyond morphological descriptions. Here, we present a complete transcriptomic analysis of the oocyst from beginning to end of its development. In addition, and to identify genes whose expression is unique to this developmental form, we compared the transcriptomes of developing oocysts with those of in vitro-derived tachyzoites and in vivo-derived bradyzoites. Our results reveal many genes whose expression is specifically up- or down-regulated in different developmental stages, including many genes that are likely critical to oocyst development, wall formation, resistance to environmental destruction and sporozoite infectivity. Of special note is the up-regulation of genes that appear “off” in tachyzoites and bradyzoites but that encode homologues of proteins known to serve key functions in those asexual stages, including a novel pairing of sporozoite-specific paralogues of AMA1 and RON2, two proteins that have recently been shown to form a crucial bridge during tachyzoite invasion of host cells. This work provides the first in-depth insight into the development and functioning of one of the most important but least studied stages in the Toxoplasma life cycle.

Stability of miRNA in human urine supports its biomarker potential

AIM miRNAs are showing utility as biomarkers in urologic disease, however, a rigorous evaluation of their stability in urine is lacking. Here, we evaluate the stability of miRNAs in urine under clinically relevant storage procedures. MATERIALS & METHODS Eight healthy individuals provided clean catch urine samples that were stored at room temperature or at 4°C for 5 days, or subjected to ten freeze-thaw cycles at -80°C. For each condition, two miRNAs, miR-16 and miR-21, were quantitated by quantitative real-time PCR. RESULTS All conditions demonstrated a surprising degree of stability of miRNAs in the urine: by the end of ten freeze-thaw cycles, 23-37% of the initial amount remained; over the 5-day period of storage at room temperature, 35% of the initial amount remained; and at 4°C, 42-56% of the initial amount remained. Both miRNAs also showed degradation at approximately the same rate. CONCLUSION miRNAs are relatively stable in urine under a variety of storage conditions, which supports their utility as urinary biomarkers.

Active Surveillance for Prostate Cancer Compared With Immediate Treatment: An Economic Analysis

The costs associated with a contemporary active surveillance strategy compared with immediate treatment for prostate cancer are not well characterized. The purpose of this study is to elucidate the health care costs of an active surveillance paradigm for prostate cancer.

Identification of tissue cyst wall components by transcriptome analysis of in vivo and in vitro Toxoplasma gondii bradyzoites.

ABSTRACT The Toxoplasma gondii bradyzoite is essential to establish persistent infection, yet little is known about what factors this developmental form secretes to establish the cyst or interact with its host cell. To identify candidate bradyzoite-secreted effectors, the transcriptomes of in vitro tachyzoites 2 days postinfection, in vitro bradyzoites 4 days postinfection, and in vivo bradyzoites 21 days postinfection were interrogated by microarray, and the program SignalP was used to identify signal peptides indicating secretion. One hundred two putative bradyzoite-secreted effectors were identified by this approach. Two candidates, bradyzoite pseudokinase 1 and microneme adhesive repeat domain-containing protein 4, were chosen for further investigation and confirmed to be induced and secreted by bradyzoites in vitro and in vivo. Thus, we report the first analysis of the transcriptomes of in vitro and in vivo bradyzoites and identify two new protein components of the Toxoplasma tissue cyst wall.

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.

The Natural History of Flare-Ups in Fibrodysplasia Ossificans Progressiva (FOP): A Comprehensive Global Assessment.

Fibrodysplasia ossificans progressiva (FOP) leads to disabling heterotopic ossification (HO) from episodic flare-ups. However, the natural history of FOP flare-ups is poorly understood. A 78-question survey on FOP flare-ups, translated into 15 languages, was sent to 685 classically-affected patients in 45 countries (six continents). Five hundred patients or knowledgeable informants responded (73%; 44% males, 56% females; ages: 1 to 71 years; median: 23 years). The most common presenting symptoms of flare-ups were swelling (93%), pain (86%), or decreased mobility (79%). Seventy-one percent experienced a flare-up within the preceding 12 months (52% spontaneous; 48% trauma-related). Twenty-five percent of those who had received an intramuscular injection reported an immediate flare-up at the injection site, 84% of whom developed HO. Axial flare-ups most frequently involved the back (41.6%), neck (26.4%), or jaw (19.4%). Flare-ups occurred more frequently in the upper limbs before 8 years of age, but more frequently in the lower limbs thereafter. Appendicular flare-ups occurred more frequently at proximal than at distal sites without preferential sidedness. Seventy percent of patients reported functional loss from a flare-up. Thirty-two percent reported complete resolution of at least one flare-up and 12% without any functional loss (mostly in the head or back). The most disabling flare-ups occurred at the shoulders or hips. Surprisingly, 47% reported progression of FOP without obvious flare-ups. Worldwide, 198 treatments were reported; anti-inflammatory agents were most common. Seventy-five percent used short-term glucocorticoids as a treatment for flare-ups at appendicular sites. Fifty-five percent reported that glucocorticoids improved symptoms occasionally whereas 31% reported that they always did. Only 12% reported complete resolution of a flare-up with glucocorticoids. Forty-three percent reported rebound symptoms within 1 to 7 days after completing a course of glucocorticoids. This study is the first comprehensive global assessment of FOP flare-ups and establishes a critical foundation for the design and evaluation of future clinical trials.

Clinical and molecular implications of mosaicism in FMR1 full mutations.

Expansions of more than 200 CGG repeats (full mutation) in the FMR1 gene give rise to fragile X syndrome (FXS) through a process that generally involves hypermethylation of the FMR1 promoter region and gene silencing, resulting in absence of expression of the encoded protein, FMRP. However, mosaicism with alleles differing in size and extent of methylation often exist within or between tissues of individuals with FXS. In the current work, CGG-repeat lengths and methylation status were assessed for eighteen individuals with FXS, including 13 mosaics, for which peripheral blood cells (PBMCs) and primary fibroblast cells were available. Our results show that for both PBMCs and fibroblasts, FMR1 mRNA and FMRP expression are directly correlated with the percent of methylation of the FMR1 allele. In addition, Full Scale IQ scores were inversely correlated with the percent methylation and positively correlated with higher FMRP expression. These latter results point toward a positive impact on cognition for full mutation mosaics with lower methylation compared to individuals with fully methylated, full mutation alleles. However, we did not observe a significant reduction in the number of seizures, nor in the severity of hyperactivity or autism spectrum disorder, among individuals with mosaic genotypes in the presentation of FXS. These observations suggest that low, but non-zero expression of FMRP may be sufficient to positively impact cognitive function in individuals with FXS, with methylation mosaicism (lowered methylation fraction) contributing to a more positive clinical outcome.

CGG allele size somatic mosaicism and methylation in FMR1 premutation alleles

Background Greater than 200 CGG repeats in the 5′UTR of the FMR1 gene lead to epigenetic silencing and lack of the FMR1 protein, causing fragile X Syndrome. Individual carriers of a premutation (PM) allele with 55–200 CGG repeats are typically unmethylated and can present with clinical features defined as FMR1-associated conditions. Methods Blood samples from 17 male PM carriers were assessed clinically and molecularly by Southern blot, western blot, PCR and QRT-PCR. Blood and brain tissue from an additional 18 PM males were also similarly examined. Continuous outcomes were modelled using linear regression and binary outcomes were modelled using logistic regression. Results Methylated alleles were detected in different fractions of blood cells in all PM cases (n=17). CGG repeat numbers correlated with percent of methylation and mRNA levels and, especially in the upper PM range, with greater number of clinical involvements. Inter-tissue/intra-tissue somatic instability and differences in percent methylation were observed between blood and fibroblasts (n=4) and also observed between blood and different brain regions in three of the 18 PM cases examined. CGG repeat lengths in lymphocytes remained unchanged over a period of time ranging from 2 to 6 years, three cases for whom multiple samples were available. Conclusions In addition to CGG size instability, individuals with a PM expanded allele can exhibit methylation and display more clinical features likely due to RNA toxicity and/or FMR1 silencing. The observed association between CGG repeat length and percent of methylation with the severity of the clinical phenotypes underscores the potential value of methylation in affected PM to further understand penetrance, inform diagnosis and expand treatment options.