Histone deacetylases suppress cgg repeat-induced neurodegeneration via transcriptional silencing in models of Fragile X Tremor Ataxia Syndrome

Fragile X Tremor Ataxia Syndrome (FXTAS) is a common inherited neurodegenerative disorder caused by expansion of a CGG trinucleotide repeat in the 59UTR of the fragile X syndrome (FXS) gene, FMR1. The expanded CGG repeat is thought to induce toxicity as RNA, and in FXTAS patients mRNA levels for FMR1 are markedly increased. Despite the critical role of FMR1 mRNA in disease pathogenesis, the basis for the increase in FMR1 mRNA expression is unknown. Here we show that overexpressing any of three histone deacetylases (HDACs 3, 6, or 11) suppresses CGG repeat-induced neurodegeneration in a Drosophila model of FXTAS. This suppression results from selective transcriptional repression of the CGG repeat-containing transgene. These findings led us to evaluate the acetylation state of histones at the human FMR1 locus. In patient-derived lymphoblasts and fibroblasts, we determined by chromatin immunoprecipitation that there is increased acetylation of histones at the FMR1 locus in pre-mutation carriers compared to control or FXS derived cell lines. These epigenetic changes correlate with elevated FMR1 mRNA expression in pre-mutation cell lines. Consistent with this finding, histone acetyltransferase (HAT) inhibitors repress FMR1 mRNA expression to control levels in pre-mutation carrier cell lines and extend lifespan in CGG repeat-expressing Drosophila. These findings support a disease model whereby the CGG repeat expansion in FXTAS promotes chromatin remodeling in cis, which in turn increases expression of the toxic FMR1 mRNA. Moreover, these results provide proof of principle that HAT inhibitors or HDAC activators might be used to selectively repress transcription at the FMR1 locus.open293

ATM and ATR protect the genome against two different types of tandem repeat instability in Fragile X premutation mice

Expansion of a tandem repeat tract is responsible for the Repeat Expansion diseases, a group of more than 20 human genetic disorders that includes those like Fragile X (FX) syndrome that result from repeat expansion in the FMR1 gene. We have previously shown that the ATM and Rad3-related (ATR) checkpoint kinase protects the genome against one type of repeat expansion in a FX premutation mouse model. By crossing the FX premutation mice to Ataxia Telangiectasia-Mutated (Atm) mutant mice, we show here that ATM also prevents repeat expansion. However, our data suggest that the ATM-sensitive mechanism is different from the ATR-sensitive one. Specifically, the effect of the ATM deficiency is more marked when the premutation allele is paternally transmitted and expansions occur more frequently in male offspring regardless of the Atm genotype of the offspring. The gender effect is most consistent with a repair event occurring in the early embryo that is more efficient in females, perhaps as a result of the action of an X-linked DNA repair gene. Our data thus support the hypothesis that two different mechanisms of FX repeat expansion exist, an ATR-sensitive mechanism seen on maternal transmission and an ATM-sensitive mechanism that shows a male expansion bias

An efficient trio-based mini-haplotyping method for genetic diagnosis of phenylketonuria

Objective: The phenylalanine hydroxylase (PAH) locus has high linkage disequilibrium. Haplotypes related to this locus may thus be considered sufficiently informative for genetic diagnosis and carrier screening using multi-allelic markers. In this study, we present an efficient method for haplotype analysis of PAH locus using multiplexing dyes. In addition, we explain how to resolve the dye shift challenge in multiplex short tandem repeat (STR) genotyping. Materials and Methods: One hundred family trios were included in this descriptive study. The forward primer of a tetra-nucleotide STR and the reverse primer of a variable number tandem repeat (VNTR) were labeled with three different non-overlapping dyes 5-carboxyfluorescein (FAM), 6-carboxy-N,N,N�,N�-tetramethylrhodamine (HEX) and 6-carboxy-N,N,N�,N�-tetramethylrhodamine (TAMRA). The polymerase chain reaction (PCR) products from each family trio were multiplexed for capillary electrophoresis and results were analyzed using Peak Scanner software. Results: Multiplexing trio products decreased the cost significantly. The TAMRA labeled products had a significant predictable shift (migrated at a slower electrophoretic rate) relative to the HEX and FAM labeled products. Through our methodology we achieve, the less inter-dye shift than intra-dye shift variance. Correcting the dye shift in the labeled products, according to the reference allele size, significantly decreased the inter-dye variability (P<0.001). Conclusion: Multiplexing trio products helps to detect and resolve the dye shift accurately in each family, which otherwise would result in diagnostic error. The dye system of FAM, HEX and TAMRA is more feasible and cheaper than other dye systems

Use of model systems to understand the etiology of fragile X-associated primary ovarian insufficiency (FXPOI)

Fragile X-associated primary ovarian insufficiency (FXPOI) is among the family of disorders caused by the expansion of a CGG repeat sequence in the 5' untranslated region of the X-linked gene FMR1. About 20% of women who carry the premutation allele (55 to 200 unmethylated CGG repeats) develop hypergonadotropic hypogonadism and cease menstruating before age 40. Some proportion of those who are still cycling show hormonal profiles indicative of ovarian dysfunction. FXPOI leads to subfertility and an increased risk of medical conditions associated with early estrogen deficiency. Little progress has been made in understanding the etiology of this clinically significant disorder. Understanding the molecular mechanisms of FXPOI requires a detailed knowledge of ovarian FMR1 mRNA and FMRP’s function. In humans, non-invasive methods to discriminate the mechanisms of the premutation on ovarian function are not available, thus necessitating the development of model systems. Vertebrate (mouse and rat) and invertebrate (Drosophila melanogaster) animal studies for the FMR1 premutation and ovarian function exist and have been instrumental in advancing our understanding of the disease phenotype. For example, rodent models have shown that FMRP is highly expressed in oocytes where it is important for folliculogenesis. The two premutation mouse models studied to date show evidence of ovarian dysfunction and, together, suggest that the long repeat in the transcript itself may have some pathological effect quite apart from any effect of the toxic protein. Further, ovarian morphology in young animals appears normal and the primordial follicle pool size does not differ from that of wild-type animals. However, there is a progressive premature decline in the levels of most follicle classes. Observations also include granulosa cell abnormalities and altered gene expression patterns. Further comparisons of these models are now needed to gain insight into the etiology of the ovarian dysfunction. Premutation model systems in non-human primates and those based on induced pluripotent stem cells show particular promise and will complement current models. Here, we review the characterization of the current models and describe the development and potential of the new models. Finally, we will discuss some of the molecular mechanisms that might be responsible for FXPOI

Use of flow cytometry in acute promyelocytic leukemia.

7075 Background: Acute promyelocytic leukemia (APL) is a highly curable disease, with the majority of failures related to hemorrhagic complications. Hence, rapid diagnosis and early initiation of therapy can drastically prevent these complications and reduce mortality. The current diagnostic strategy utilizing fluorescent in situ hybridization (FISH) is often time-consuming and not readily available in some institutions. This is a review of our institutional experience with the use flow cytometry for diagnosis of APL. Methods: All cases with t(15;17) by FISH and karyotype between 2006 and 2012 were identified. A second group of consecutive cases of non-M3 acute myeloid leukemia (AML) and negative FISH and karyotype for t(15;17) was used for comparison. A total of 21 APL and 42 non-M3 AML cases were analyzed. Results: Both groups were comparable in regard to age, gender, white blood cell count, hemoglobin, blast count and lactate dehydrogenase level. The APL group had significantly higher prevalence of thrombocytopenia, disseminated intravascular coagulation and clinical bleeding at time of admission. Expression of CD11c was lacking in 92.3% (12/13) of APL and 14.6% (6/41) of non-M3 AML cases (p&lt;0.0001). CD34 expression was lacking in 68.4% (13/19) of APL and 42.9% (18/42) of non-M3 AML cases (p=0.06). HLA-DR expression was lacking in 88.9% (16/18) of APL and 9.5% (4/42) of non-M3 AML cases (p&lt;0.0001). Given that APL prevalence is 5% among all AML cases, lack of expression of CD11c, CD34 and HLA-DR have negative predictive values (NPVs) of 99.5%, 97.1% and 9933%, respectively. Among various immunophenotypic profiles, lack of expression of CD11c had the highest NPV (99.5%, p&lt;0.0001) and simultaneous lack of expression of CD11c and HLA-DR had the highest specificity (95.1%, p&lt;0.0001). Conclusions: Expression of CD11c reliably excludes the diagnosis of APL in the majority of AML cases. Flow cytometry has the potential of replacing FISH as the initial test to prompt initiating therapy in APL patients. </jats:p