DM1 Phenotype Variability and Triplet Repeat Instability: Challenges in the Development of New Therapies.

Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disease caused by an unstable cardiotocography (CTG) repeat expansion in the DMPK gene. This disease is characterized by high clinical and genetic variability, leading to some difficulties in the diagnosis and prognosis of DM1. Better understanding the origin of this variability is important for developing new challenging therapies and, in particular, for progressing on the path of personalized treatments. Here, we reviewed CTG triplet repeat instability and its modifiers as an important source of phenotypic variability in patients with DM1.

Human mismatch repair protein hMutLα is required to repair short slipped-DNAs of trinucleotide repeats.

Mismatch repair (MMR) is required for proper maintenance of the genome by protecting against mutations. The mismatch repair system has also been implicated as a driver of certain mutations, including disease-associated trinucleotide repeat instability. We recently revealed a requirement of hMutSß in the repair of short slip-outs containing a single CTG repeat unit (1). The involvement of other MMR proteins in short trinucleotide repeat slip-out repair is unknown. Here we show that hMutLα is required for the highly efficient in vitro repair of single CTG repeat slip-outs, to the same degree as hMutSß. HEK293T cell extracts, deficient in hMLH1, are unable to process single-repeat slip-outs, but are functional when complemented with hMutLα. The MMR-deficient hMLH1 mutant, T117M, which has a point mutation proximal to the ATP-binding domain, is defective in slip-out repair, further supporting a requirement for hMLH1 in the processing of short slip-outs and possibly the involvement of hMHL1 ATPase activity. Extracts of hPMS2-deficient HEC-1-A cells, which express hMLH1, hMLH3, and hPMS1, are only functional when complemented with hMutLα, indicating that neither hMutLß nor hMutLγ is sufficient to repair short slip-outs. The resolution of clustered short slip-outs, which are poorly repaired, was partially dependent upon a functional hMutLα. The joint involvement of hMutSß and hMutLα suggests that repeat instability may be the result of aberrant outcomes of repair attempts.

The role of genetic and autoimmune factors in premature ovarian failure.

PURPOSE: To identify the role of both genetic (number of CGG repeats in the FMR1 gene) and autoimmune factors (anti-ovarian antibodies) in premature ovarian failure (POF). METHODS: In cross-sectional study, 78 women with POF were divided into 3 groups by the number of CGG repeats (less than 28, 28-36, more than 36) in any of the FMR1 gene alleles. We performed the detection of skewed X-chromosome inactivation, CGG repeats in the FMR1 gene, anti-ovarian antibodies (AOA) and sex hormones tests. RESULTS: Compared to a higher or lower number of CGG repeats the 28-36 triple CGG counts are strongly associated with the AOA detection (RR = 19.23, 95% CI = 2.63-100.0). The women with autoimmune-driven POF have significantly higher anti-Mullerian hormone levels in comparison to women with non-autoimmune-driven POF. CONCLUSION: The presence of AOA above 10 IU/mL is associated with the normal number of CGG repeats in regard to ovarian reserve and a better preservation of follicular primordial pool in the women with POF.

RGG repeats of PrP-like Shadoo protein bind nucleic acids.

Shadoo (Sho) is a central nervous system glycoprotein with characteristics similar to those of the cellular prion protein PrP(C), each containing a highly conserved hydrophobic domain (HD) and an N-terminal repeat region. Whereas PrP(C) includes histidine-containing octarepeats, the Sho region N-terminal to the HD includes tandem positively charged "RGG boxes", predicted to bind RNA. Here, we demonstrate that Sho binds DNA and RNA in vitro via this arginine-rich region.

Polypurine-repeat-containing RNAs: a novel class of long non-coding RNA in mammalian cells.

In higher eukaryotic cells, long non-protein-coding RNAs (lncRNAs) have been implicated in a wide array of cellular functions. Cell- or tissue-specific expression of lncRNA genes encoded in the mammalian genome is thought to contribute to the complex gene networks needed to regulate cellular function. Here, we have identified a novel species of polypurine triplet repeat-rich lncRNAs, designated as GAA repeat-containing RNAs (GRC-RNAs), that localize to numerous punctate foci in the mammalian interphase nuclei. GRC-RNAs consist of a heterogeneous population of RNAs, ranging in size from ~1.5 kb to ~4 kb and localize to subnuclear domains, several of which associate with GAA.TTC-repeat-containing genomic regions. GRC-RNAs are components of the nuclear matrix and interact with various nuclear matrix-associated proteins. In mitotic cells, GRC-RNAs form distinct cytoplasmic foci and, in telophase and G1 cells, localize to the midbody, a structure involved in accurate cell division. Differentiation of tissue culture cells leads to a decrease in the number of GRC-RNA nuclear foci, albeit with an increase in size as compared with proliferating cells. Conversely, the number of GRC-RNA foci increases during cellular transformation. We propose that nuclear GRC-RNAs represent a novel family of mammalian lncRNAs that might play crucial roles in the cell nucleus.

Myotonic dystrophy type 1-associated CTG repeats disturb the expression and subcellular distribution of microtubule-associated proteins MAP1A, MAP2, and MAP6/STOP in PC12 cells.

To study the effect of DM1-associated CTG repeats on neuronal function, we developed a PC12 cell-based model that constitutively expresses the DMPK gene 3'-untranslated region with 90 CTG repeats (CTG90 cells). As CTG90 cells exhibit impaired neurite outgrowth and as microtubule-associated proteins (MAPs) are crucial for microtubule stability, we analyzed whether MAPs are a target of CTG repeats. NGF induces mRNA expression of Map2, Map1a and Map6 in control cells (PC12 cells transfected with the empty vector), but this induction is abolished for Map2 and Map1a in CTG90 cells. MAP2 and MAP6/STOP proteins decrease in NGF-treated CTG90 cells, whereas MAP1A increases. Data suggest that CTG repeats might alter somehow the expression of MAPs, which appears to be related with CTG90 cell-deficient neurite outgrowth. Decreased MAP2 levels found in the hippocampus of a DM1 mouse model indicates that targeting of MAPs expression by CTG repeats might be relevant to DM1.

Complementary roles for exonuclease 1 and Flap endonuclease 1 in maintenance of triplet repeats.

Trinucleotide repeats can form stable secondary structures that promote genomic instability. To determine how such structures are resolved, we have defined biochemical activities of the related RAD2 family nucleases, FEN1 (Flap endonuclease 1) and EXO1 (exonuclease 1), on substrates that recapitulate intermediates in DNA replication. Here, we show that, consistent with its function in lagging strand replication, human (h) FEN1 could cleave 5'-flaps bearing structures formed by CTG or CGG repeats, although less efficiently than unstructured flaps. hEXO1 did not exhibit endonuclease activity on 5'-flaps bearing structures formed by CTG or CGG repeats, although it could excise these substrates. Neither hFEN1 nor hEXO1 was affected by the stem-loops formed by CTG repeats interrupting duplex regions adjacent to 5'-flaps, but both enzymes were inhibited by G4 structures formed by CGG repeats in analogous positions. Hydroxyl radical footprinting showed that hFEN1 binding caused hypersensitivity near the flap/duplex junction, whereas hEXO1 binding caused hypersensitivity very close to the 5'-end, correlating with the predominance of hFEN1 endonucleolytic activity versus hEXO1 exonucleolytic activity on 5'-flap substrates. These results show that FEN1 and EXO1 can eliminate structures formed by trinucleotide repeats in the course of replication, relying on endonucleolytic and exonucleolytic activities, respectively. These results also suggest that unresolved G4 DNA may prevent key steps in normal post-replicative DNA processing.

Relevance of triple CGG repeats in the FMR1 gene to ovarian reserve.

Most individuals demonstrate 29-30 CGG triple repeats on the FMR1 gene. This may functionally represent a normal range in regard to ovarian reserve. Higher counts reflect risk towards premature ovarian senescence, but lower counts have not been investigated before and, therefore, were the principal subject of this investigation. Amongst 316 consecutive infertility patients, 94 demonstrated <28 repeats (group A), 163 28-33 repeats (group B, considered normal repeat numbers) and 59 > or =34 repeats (group C). The three groups did not differ in age, FSH or anti-Müllerian hormone (AMH) concentrations. Neither FSH nor AMH correlated in linear regression with <28 CGG repeats. In logistic regression, AMH of < or =0.8 ng/ml (indicative of diminished ovarian reserve at all ages) was, however, significantly associated with number of repeats (P < 0.001). Every decrease by five CGG repeats in group A increased the likelihood of diminished ovarian reserve by 40%, while every increase by five CGG repeats in group C increased risk by 50% (both P < 0.002). AMH of < or =0.8 ng/ml statistically correlated overall with decreasing triple CGG repeats throughout all ranges (P < 0.001). Approximately 29-30 CGG repeats appear reflective of normal ovarian reserve, with higher and lower counts denoting similar risks towards premature ovarian senescence.

The androgen receptor CAG repeat length polymorphism associates with prostate volume in Finnish men with benign prostatic hyperplasia.

The development of benign prostatic hyperplasia requires the presence of testicular androgens during prostate development, puberty, and ageing. We thus examined the association of three polymorphisms, namely, CYP3A5 6986A>G, CYP19A1 1531C>T, and androgen receptor (AR) gene CAG repeat length, which have previously been linked to the androgen pathway and with clinical characteristics of benign prostatic hyperplasia. Tissue samples from 262 consecutive prostate operations were used for genotyping. Prostate volumes and prostate-specific antigen values were collected from patient records. Linear regression analysis was performed to study the polymorphisms in an age-adjusted model. We did not find any association between the CYP3A5 6986A>G polymorphism and clinical characteristics of benign prostatic hyperplasia. Further, the previously published CYP19A1 1531C>T polymorphism association with an enlarged prostate could not be confirmed with this material. However, we detected an association between short AR gene CAG repeat length and a small prostate volume, which confirms a previous finding in the Finnish population. The data presented suggest a negligible role for the CYP3A5 6986A>G polymorphism in benign prostate enlargement in the Finnish population. However, the results presented do provide further evidence for potentially different genetic mechanisms behind benign prostatic hyperplasia in Finnish and other Caucasian populations. This is based on the conflicting results for AR gene CAG repeat length associations with benign prostatic hyperplasia found in published works.

A patient-derived cellular model for Huntington's disease reveals phenotypes at clinically relevant CAG lengths.

The huntingtin protein participates in several cellular processes that are disrupted when the polyglutamine tract is expanded beyond a threshold of 37 CAG DNA repeats in Huntington's disease (HD). Cellular biology approaches to understand these functional disruptions in HD have primarily focused on cell lines with synthetically long CAG length alleles that clinically represent outliers in this disease and a more severe form of HD that lacks age onset. Patient-derived fibroblasts are limited to a finite number of passages before succumbing to cellular senescence. We used human telomerase reverse transcriptase (hTERT) to immortalize fibroblasts taken from individuals of varying age, sex, disease onset, and CAG repeat length, which we have termed TruHD cells. TruHD cells display classic HD phenotypes of altered morphology, size and growth rate, increased sensitivity to oxidative stress, aberrant adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratios, and hypophosphorylated huntingtin protein. We additionally observed dysregulated reactive oxygen species (ROS)-dependent huntingtin localization to nuclear speckles in HD cells. We report the generation and characterization of a human, clinically relevant cellular model for investigating disease mechanisms in HD at the single-cell level, which, unlike transformed cell lines, maintains functions critical for huntingtin transcriptional regulation and genomic integrity.

Male infertility and variation in CAG repeat length in the androgen receptor gene: a meta-analysis.

CONTEXT: Many studies have investigated the association between male infertility and trinucleotide repeat polymorphisms in the androgen receptor (AR) gene, but no comprehensive meta-analysis of all published studies has been conducted. OBJECTIVE: Our goals were to summarize published data on associations between AR CAG and GGC repeat lengths and male infertility and investigate sources of variation between study results. DATA SOURCES: We searched for reports published before October 2006 using Medline, PubMed, and Web of Science. STUDY SELECTION: All selected studies included the following: a case group with infertility as measured by semen parameters, a control group of known or presumed fertile men, and measurement of CAG and/or GGC repeat lengths among cases and controls. Thirty-nine reports were selected based on these criteria, and 33 were ultimately included in the meta-analysis. DATA EXTRACTION: One investigator extracted data on sample size, mean and sd of trinucleotide repeat length, and study characteristics. DATA SYNTHESIS: Estimates of the standardized mean difference (95% confidence interval) were 0.19 (0.09-0.29) for the 33 studies and 0.31 (0.14-0.47) for a subset of 13 studies that used more stringent case and control selection criteria. Thus, in both groups, cases had statistically significantly longer CAG repeat length than controls. Publication date appeared to be a significant source of variation between studies. CONCLUSIONS: This meta-analysis provides support for an association between increased androgen receptor CAG length and idiopathic male infertility, suggesting that even subtle disruptions in the androgen axis may compromise male fertility.

Androgen receptor CAG and GGN repeat polymorphisms influence performance in boys and girls.

BACKGROUND: Shorter CAG and GGN androgen receptor (AR) repeat polymorphisms are associated with stronger androgen signaling, and therefore, could influence lean mass and exercise performance during growth. METHODS: Physical fitness and body composition were measured by standardized procedures and the length of CAG and GGN repeats was determined by PCR and fragment analysis in 152 boys (11.5±2.6 years; Tanner ≤5) and 116 girls (10.1±3.2 years; Tanner ≤5). Individuals were grouped as CAG short (CAGS) if harboring repeat lengths of ≤21 and CAG long (CAGL) if CAG >21. Moreover, subjects were grouped as GGN short (GGNS) if harboring repeat lengths of ≤23 and GGN long (GGNL) if GGN>23. RESULTS: No significant differences in anthropometrics and body composition were observed between either CAGS and CAGL groups and GGNS and GGNL groups. Boys harboring CAGS completed the 300-meter test faster than their CAGL counterparts. Moreover, girls from the GGNL group showed a significant higher VO2max than those in the GGNS group. CONCLUSIONS: In summary, carrying a short allele of the androgen receptor CAG repeat polymorphism is associated to higher anaerobic performance in boys, whereas long alleles of androgen receptor GGN polymorphisms are associated to higher aerobic capacity in girls.

Nuclear targeting of the CaMKII anchoring protein alphaKAP is regulated by alternative splicing and protein kinases.

alphaKAP is an anchoring protein for the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and is encoded within the same gene as the CaMKIIalpha isoform. alphaKAP co-assembles with CaMKII and targets such heteromers to the membrane of the sarcoplasmic reticulum, where CaMKII can regulate Ca(2+) homeostasis. CaMKII has also nuclear functions in skeletal muscle, however, the nuclear targeting mechanism has been elusive. We show here that developmentally regulated splicing of exon Ealpha(B) generates a functional nuclear localization signal (NLS) in alphaKAP(B), the dominant alphaKAP variant in mature muscle. The alphaKAP(A) variant lacks the NLS and dominates in developing muscle before and around birth. Both alphaKAP variants localize to membranes, but a small fraction of alphaKAP(B) is additionally found in the nucleus. Indeed, alpha-karyopherins that mediate nuclear import bound to alphaKAP(B) but not alphaKAP(A) in vitro. When the N-terminal membrane anchor of alphaKAP was deleted, localization of alphaKAP(B) but not alphaKAP(A) became predominantly nuclear. Co-expression of constitutively active CaMKI and IV, which do not bind to alphaKAP, interfered with nuclear localization of alphaKAP(B). CaMKIIalpha was found essentially exclusively in the cytoplasm when expressed in cell lines but was targeted to the nucleus when co-expressed with the nuclear form of alphaKAP(B). Thus, nuclear targeting of cytoplasmic CaMKII isoforms by alphaKAP may be regulated by developmentally controlled alternative splicing and by protein kinases.

Transcription and nuclear transport of CAG/CTG trinucleotide repeats in yeast.

Trinucleotide repeats are involved in several neurological disorders in humans. DNA sequences containing CAG/CTG repeats are prone to slippage during replication and double-strand break repair. The effects of trinucleotide repeats on transcription and on nuclear export were analyzed in vivo in yeast. Transcription of a CAG/CTG trinucleotide repeat in the 3'-untranslated region of a URA3 reporter gene leads to transcription of messenger RNAs several kilobases longer than the expected size. These long mRNAs form more readily when CAG rather than CTG repeats are transcribed. CAG- or CUG-containing transcripts show a non-homogeneous cellular localization. We propose that long mRNAs result from transcription slippage, and discuss the possible implications for human diseases.

CUG repeat binding protein (CUGBP1) interacts with the 5' region of C/EBPbeta mRNA and regulates translation of C/EBPbeta isoforms.

The transcription factor CCAAT/enhancer binding protein beta, C/EBPbeta, plays a significant role in the regulation of hepatocyte growth and differentiation. A single mRNA coding for C/EBPbeta produces several protein isoforms. Two pathways for generation of low molecular weight C/EBPbeta isoforms have been described: specific proteolytic cleavage and initiation of translation from different AUG codons of C/EBPbeta mRNA. A truncated C/EBPbeta isoform, LIP, is induced in rat livers in response to partial hepatectomy (PH) via the alternative translation mechanism. Here we present evidence that CUG repeat binding protein, CUGBP1, interacts with the 5' region of C/EBPbeta mRNA and regulates translation of C/EBPbeta isoforms. Two binding sites for CUGBP1 are located side by side between the first and second AUG codons of C/EBPbeta mRNA. One binding site is observed in an out of frame short open reading frame (sORF) that has been previously shown to regulate initiation of translation from different AUG codons of C/EBPbeta mRNA. Analysis of cytoplasmic and polysomal proteins from rat liver after PH showed that CUGBP1 is associated with polysomes that translate low molecular weight isoforms of C/EBPbeta. The binding activity of CUGBP1 to the 5' region of C/EBPbeta mRNA shows increased association with these polysomal fractions after PH. Addition of CUGBP1 into a cell-free translation system leads to increased translation of low molecular weight isoforms of C/EBPbeta. Our data demonstrate that CUGBP1 protein is an important component for the regulation of initiation from different AUG codons of C/EBPbeta mRNA.

[Features of interaction of complexes cortisol-apolipoprotein A-I and tetrahydrocortisol-apolipoprotein A-I with eukariotic DNA].

On primary culture of hepatocytes it is shown, that a complex cortisol-apolipoprotein A-I did not change rate of biosynthesis DNA and protein, whereas the complex tetrahydrocortisol-apolipoprotein A-I (THC-apoA-I) essentially raised rate of incorporation 3H-thymidine in DNA and 14C-leucine into protein. By a method of small-angle X-ray scattering it is shown, that appreciable interaction with eukariotic DNA is marked only in case of use of a complex THC-apoA-I, thus there is local fusion of DNA. The most probable region of interaction of the given complex with DNA is repetition (GCC)n the type, included in structure of many genes eukariot, including the human. It is synthesized oligonucleotid (duplex) of this type. It is shown, that at his interaction with complex THC-apoA-I there is a formation of more difficult complex, which breaks up with formation of complementary chains of oligonucleotides. The last also enter interaction with complex THC-apoA-I. It is given of kinetic this multiphasic process. Interaction of a complex cortisol-anoA-I with a duplex is less specific and does not result reduce in decay of the duplex and in formation of complementary oligonucleotides.

Identification of ter94, Drosophila VCP, as a modulator of polyglutamine-induced neurodegeneration.

We have successfully generated a Drosophila model of human polyglutamine (polyQ) diseases by the targeted expression of expanded-polyQ (ex-polyQ) in the Drosophila compound eye. The resulting eye degeneration is progressive and ex-polyQ dosage- and ex-polyQ length-dependent. Furthermore, intergenerational changes in repeat length were observed in homozygotes, with concomitant changes in the levels of degeneration. Through genetic screening, using this fly model, we identified loss-of-function mutants of the ter94 gene that encodes the Drosophila homolog of VCP/CDC48, a member of the AAA+ class of the ATPase protein family, as dominant suppressors. The suppressive effects of the ter94 mutants on ex-polyQ-induced neurodegeneration correlated well with the degrees of loss-of-function, but appeared not to result from the inhibition of ex-polyQ aggregate formation. In the ex-polyQ-expressing cells of the late pupa, an upregulation of ter94 expression was observed prior to cell death. Co-expression of ter94 with ex-polyQ severely enhanced eye degeneration. Interestingly, when ter94 was overexpressed in the eye by increasing the transgene copies, severe eye degeneration was induced. Furthermore, genetical studies revealed that ter94 was not involved in grim-, reaper-, hid-, ced4-, or p53-induced cell death pathways. From these observations, we propose that VCP is a novel cell death effector molecule in ex-polyQ-induced neurodegeneration, where the amount of VCP is critical. Control of VCP expression may thus be a potential therapeutic target in ex-polyQ-induced neurodegeneration.

Identification of RTG2 as a modifier gene for CTG*CAG repeat instability in Saccharomyces cerevisiae.

Trinucleotide repeats (TNRs) undergo frequent mutations in families affected by TNR diseases and in model organisms. Much of the instability is conferred in cis by the sequence and length of the triplet tract. Trans-acting factors also modulate TNR instability risk, on the basis of such evidence as parent-of-origin effects. To help identify trans-acting modifiers, a screen was performed to find yeast mutants with altered CTG.CAG repeat mutation frequencies. The RTG2 gene was identified as one such modifier. In rtg2 mutants, expansions of CTG.CAG repeats show a modest increase in rate, depending on the starting tract length. Surprisingly, contractions were suppressed in an rtg2 background. This creates a situation in a model system where expansions outnumber contractions, as in humans. The rtg2 phenotype was apparently specific for CTG.CAG repeat instability, since no changes in mutation rate were observed for dinucleotide repeats or at the CAN1 reporter gene. This feature sets rtg2 mutants apart from most other mutants that affect genetic stability both for TNRs and at other DNA sequences. It was also found that RTG2 acts independently of its normal partners RTG1 and RTG3, suggesting a novel function of RTG2 that helps modify CTG.CAG repeat mutation risk.

Polymorphic CUG repeats in human mRNAs and their effects on gene expression.

Expanded CUG repeats in the 3'-untranslated region (UTR) of the gene encoding myotonic dystrophy protein kinase (DMPK) cause myotonic dystrophy type 1 disease (DM1). The presence of such repeats has been found to impede gene expression at several levels in model systems. We took a bioinformatic approach to survey all human mRNA sequences for polymorphic CUG repeats. Our survey revealed that CUG repeats occur widely in various regions of mRNAs, with higher frequency in protein coding regions than 5'-UTRs or 3'-UTRs. About 30 genes were found to contain CUG repeats that are polymorphic in the number of repeats, suggesting the potential to expand or shrink. However, long polymorphic repeats were restricted to the 3'-UTR of the DMPK gene and the coding region of the ribosomal protein L14 gene. Using cell-free translation systems, we showed that extended CUG repeats can inhibit protein synthesis in vitro in the rabbit reticulocyte lysate, but not in wheat germ extracts, consistent with our previous finding of an interaction of CUG repeats with the protein kinase PKR. In transfected cells, CUG repeats can inhibit gene expression both in cis and in trans. However, observations with PKR-minus cells indicate that these effects are not primarily attributable to the interaction of extended CUG repeats with PKR. Northwestern blotting detected the presence in human cells of more CUG-binding proteins than are currently known.