Genetic determinants of normal variation in coagulation factor (F) IX levels: genome-wide scan and examination of the FIX structural gene.

BACKGROUND: High-normal and elevated plasma FIX activity (FIX:C) levels are associated with increased risk for venous- and possibly arterial-thrombosis. OBJECTIVE: Because the broad normal range for FIX:C involves a substantial unknown genetic component, we sought to identify quantitative-trait loci (QTLs) for this medically important hemostasis trait. METHODS: We performed a genome-wide screen and a resequencing-based variation scan of the known functional regions of every distinct FIX gene (F9) in the genetic analysis of idiopathic thrombophilia project (GAIT), a collection of 398 Spanish-Caucasians from 21 pedigrees. RESULTS: We found no evidence for linkage (LOD scores <1.5) despite genotyping more than 540 uniformly-spaced microsatellites. We identified 27 candidate F9 polymorphisms, including three in cis-elements responsible for the increase in FIX:C that occurs with aging, but found no significant genotype-specific differences in mean FIX:C levels (P-values > or = 0.11) despite evaluating every polymorphism in GAIT by marginal multicovariate measured-genotype association analysis. CONCLUSIONS: The heritable component of interindividual FIX:C variability likely involves a collection of QTLs with modest effects that may reside in genes other than F9. Nevertheless, because the alleles of these 27 polymorphisms exhibited a low overall degree of linkage disequilibrium, we are currently defining their haplotypes to interrogate several highly-conserved non-exonic sequences and other F9 segments not examined here.

Come FLY with us: toward understanding fragile X syndrome.

The past few years have seen an increased number of articles using Drosophila as a model system to study fragile X syndrome. Phenotypic analyses have demonstrated an array of neuronal and behavioral defects similar to the phenotypes reported in mouse models as well as human patients. The availability of both cellular and molecular tools along with the power of genetics makes the tiny fruit fly a premiere model in elucidating the molecular basis of fragile X syndrome. Here, we summarize the advances made in recent years in the characterization of fragile X Drosophila models and the identification of new molecular partners in neural development.

Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function.

Loss of fragile X mental retardation protein (FMRP) function causes the fragile X mental retardation syndrome. FMRP harbors three RNA binding domains, associates with polysomes, and is thought to regulate mRNA translation and/or localization, but the RNAs to which it binds are unknown. We have used RNA selection to demonstrate that the FMRP RGG box binds intramolecular G quartets. This data allowed us to identify mRNAs encoding proteins involved in synaptic or developmental neurobiology that harbor FMRP binding elements. The majority of these mRNAs have an altered polysome association in fragile X patient cells. These data demonstrate that G quartets serve as physiologically relevant targets for FMRP and identify mRNAs whose dysregulation may underlie human mental retardation.

Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome.

Fragile X syndrome results from the absence of the RNA binding FMR protein. Here, mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. We identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. We conclude that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and we identify candidate genes relevant to this phenotype.

The female and the fragile X reviewed.

Nearly 15 years ago, female carriers of the fragile X mental retardation syndrome were noted to have an increased incidence of twin pregnancies. Since then, much evidence has accumulated supporting the notion of ovarian dysfunction in fragile X carriers, in the forms of increased dizygotic twinning and premature ovarian failure. However, despite a decade and a half of research regarding this association, the underlying mechanism remains a mystery. This article reviews the population-based studies that have examined this association and discusses possible reasons for the variations in results. In addition, results from more recent studies on endocrine function in fragile X carriers are discussed. These data, when considered in conjunction with our emerging understanding of the molecular biology of the fragile X gene (FMR1) and its protein product (FMRP), are beginning to elucidate possible mechanisms for the association between fragile X syndrome and ovarian dysfunction.

Reduced FMRP and increased FMR1 transcription is proportionally associated with CGG repeat number in intermediate-length and premutation carriers.

The 5' untranslated CGG repeat in the fragile X mental retardation-1 (FMR1) gene is expanded in families with fragile X syndrome, with more than 200 CGGs resulting in mental retardation due to the absence of the encoded fragile X mental retardation protein (FMRP). Intermediate and premutation alleles, containing between approximately 40 and 200 repeats, express grossly normal FMRP levels and such carriers are widely believed to be non-penetrant, despite continued reports of subtle cognitive/psychosocial impairment and other phenotypes. Using a highly sensitive quantification assay, we demonstrate significantly diminished FMRP levels in carriers, negatively correlated with repeat number. Despite reduced FMRP, these carrier alleles overexpress FMR1, resulting in a positive correlation between repeat number and FMR1 message level. These biochemical deviations associated with intermediate and premutation FMR1 alleles, found in approximately 4% of the population, suggest that the phenotypic spectrum of fragile X syndrome may need to be revisited.

The fragile X mental retardation protein inhibits translation via interacting with mRNA.

Fragile X syndrome is a frequent form of inherited mental retardation caused by functional loss of the fragile X mental retardation protein, FMRP. The function of FMRP is unknown, as is the mechanism by which its loss leads to cognitive deficits. Recent studies have determined that FMRP is a selective RNA-binding protein associated with polyribosomes, leading to the hypothesis that FMRP may be involved in translational regulation. Here we show that purified recombinant FMRP causes a dose-dependent translational inhibition of brain poly(A) RNA in rabbit reticulocyte lysate without accelerated mRNA degradation. In our translation reaction FMRP interacts with other messenger ribonucleoproteins and pre-exposure of FMRP to mRNA significantly increased the potency of FMRP as a translation inhibitor. Translation suppression by FMRP is reversed in a trans-acting manner by the 3'-untranslated portion of the Fmr1 message, which binds FMRP, suggesting that FMRP inhibits translation via interacting with mRNA. Consistently FMRP suppresses translation of the parathyroid hormone transcript, which binds FMRP, but not the beta-globin transcript, which does not bind FMRP. Moreover, removing the FMRP-binding site on a translation template abolishes the inhibitory effect of FMRP. Taken together, our results support the hypothesis that FMRP inhibits translation via interactions with the translation template.

TMS1, a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast cancers.

Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration that serves as an alternative to genetic defects in the inactivation of tumor suppressor and other genes in human cancers. The hypothesis that aberrant methylation plays a direct causal role in carcinogenesis hinges on the question of whether aberrant methylation is sufficient to drive gene silencing. To identify downstream targets of methylation-induced gene silencing, we used a human cell model in which aberrant CpG island methylation is induced by ectopic expression of DNA methyltransferase. Here we report the isolation and characterization of TMS1 (target of methylation-induced silencing), a novel CpG island-associated gene that becomes hypermethylated and silenced in cells overexpressing DNA cytosine-5-methyltransferase-1. We also show that TMS1 is aberrantly methylated and silenced in human breast cancer cells. Forty percent (11 of 27) of primary breast tumors exhibited aberrant methylation of TMS1. TMS1 is localized to chromosome 16p11.2-12.1 and encodes a 22-kDa predicted protein containing a COOH-terminal caspase recruitment domain, a recently described protein interaction motif found in apoptotic signaling molecules. Ectopic expression of TMS1 induced apoptosis in 293 cells and inhibited the survival of human breast cancer cells. The data suggest that methylation-mediated silencing of TMS1 confers a survival advantage by allowing cells to escape from apoptosis, supporting a new role for aberrant methylation in breast tumorigenesis.

Fragile X CGG repeat structures among African-Americans: identification of a novel factor responsible for repeat instability.

The cryptic CGG repeat responsible for the fragile X syndrome, located in the 5'-UTR of FMR1, is unique compared with the many other triplet repeat-causing diseases, making it ideal for identifying factors involved in repeat expansion that may be common to other triplet repeat diseases. To date, a number of factors have been identified which may influence repeat instability, including the number and position of interspersed AGGs, length of the 3' pure CGG repeat and haplotype background. However, nearly all such data were derived from studies of Caucasians. Using a large African-American population, we present the only comprehensive examination of factors associated with CGG repeat instability in a non-Caucasian population. Among Caucasians, susceptible alleles were thought to come from those in the intermediate repeat range (41-60 repeats); however, we find that susceptible alleles may come from a larger repeat pool (35-60 repeats) and are better defined by their pure CGG repeat and/or -presence of only one AGG interruption. These results demonstrate the existence of different susceptible alleles among world populations and may account for the similar prevalence of the fragile X syndrome in African-Americans compared with Caucasians despite the lower frequency of inter-mediate sized alleles in the African-American population. Finally, we show that repeat structures among unaffected African-Americans with the most frequent fragile X haplotype background are either pure or contain a single distal interruption. We propose that the lack of a proximal most interruption is a novel factor involved in CGG repeat instability.

Association of the mouse infertility factor DAZL1 with actively translating polyribosomes.

The DAZ (Deleted in AZoospermia) gene family was isolated from a region of the human Y chromosome long arm that is deleted in about 10% of infertile men with idiopathic azoospermia. DAZ and an autosomal DAZ-like gene, DAZL1, are expressed in germ cells only. They encode proteins with an RNA recognition motif and with either a single copy (in DAZL1) or multiple copies (in DAZ) of a DAZ repeat. A role for DAZL1 and DAZ in spermatogenesis is supported by their homology to a Drosophila male infertility protein Boule and by sterility of Dazl1 knock-out mice. The biological function of these proteins remains unknown. We found that DAZL1 and DAZ bound similarly to various RNA homopolymers in vitro. We also used an antibody against the human DAZL1 to determine the subcellular localization of DAZL1 in mouse testis. The sedimentation profiles of DAZL1 in sucrose gradients indicate that DAZL1 is associated with polyribosomes, and further capture of DAZL1 on oligo(dT) beads demonstrates that the association is mediated through the binding of DAZL1 to poly(A) RNA. Our results suggest that DAZL1 is involved in germ-cell specific regulation of mRNA translation.

Understanding the molecular basis of fragile X syndrome.

Fragile X syndrome, a common form of inherited mental retardation, is mainly caused by massive expansion of CGG triplet repeats located in the 5'-untranslated region of the fragile X mental retardation-1 ( FMR1 ) gene. In patients with fragile X syndrome, the expanded CGG triplet repeats are hypermethylated and the expression of the FMR1 gene is repressed, which leads to the absence of FMR1 protein (FMRP) and subsequent mental retardation. FMRP is an RNA-binding protein that shuttles between the nucleus and cytoplasm. This protein has been implicated in protein translation as it is found associated with polyribosomes and the rough endoplasmic reticulum. We discuss here the recent progress made towards understanding the molecular mechanism of CGG repeat expansion and physiological function(s) of FMRP. These studies will not only help to illuminate the molecular basis of the general class of human diseases with trinucleotide repeat expansion but also provide an avenue to understand aspects of human cognition and intelligence.

(Over)correction of FMR1 deficiency with YAC transgenics: behavioral and physical features.

Fragile X syndrome is a common cause of mental retardation involving loss of expression of the FMR1 gene. The role of FMR1 remains undetermined but the protein appears to be involved in RNA metabolism. Fmr1 knockout mice exhibit a phenotype with some similarities to humans, such as macroorchidism and behavioral abnormalities. As a step toward understanding the function of FMR1 and the determination of the potential for therapeutic approaches to fragile X syndrome, yeast artificial chromosome (YAC) transgenic mice were generated in order to determine whether the Fmr1 knockout mouse phenotype could be rescued. Several transgenic lines were generated that carried the entire FMR1 locus with extensive amounts of flanking sequence. We observed that the YAC transgene supported production of the human protein (FMRP) which was present at levels 10 to 15 times that of endogenous protein and was expressed in a cell- and tissue-specific manner. Macro-orchidism was absent in knockout mice carrying the YAC transgene indicating functional rescue by the human protein. Given the complex behavioral phenotype in fragile X patients and the mild phenotype previously reported for the Fmr1 knockout mouse, we performed a more thorough evaluation of the Fmr1 knockout phenotype using additional behavioral assays that had not previously been reported for this animal model. The mouse displayed reduced anxiety-related responses with increased exploratory behavior. FMR1 YAC transgenic mice overexpressing the human protein did produce opposing behavioral responses and additional abnormal behaviors were also observed. These findings have significant implications for gene therapy for fragile X syndrome since overexpression of the gene may harbor its own phenotype.

Survey of the fragile X syndrome CGG repeat and the short-tandem-repeat and single-nucleotide-polymorphism haplotypes in an African American population.

Previous studies have shown that specific short-tandem-repeat (STR) and single-nucleotide-polymorphism (SNP)-based haplotypes within and among unaffected and fragile X white populations are found to be associated with specific CGG-repeat patterns. It has been hypothesized that these associations result from different mutational mechanisms, possibly influenced by the CGG structure and/or cis-acting factors. Alternatively, haplotype associations may result from the long mutational history of increasing instability. To understand the basis of the mutational process, we examined the CGG-repeat size, three flanking STR markers (DXS548-FRAXAC1-FRAXAC2), and one SNP (ATL1) spanning 150 kb around the CGG repeat in unaffected (n=637) and fragile X (n=63) African American populations and compared them with unaffected (n=721) and fragile X (n=102) white populations. Several important differences were found between the two ethnic groups. First, in contrast to that seen in the white population, no associations were observed among the African American intermediate or "predisposed" alleles (41-60 repeats). Second, two previously undescribed haplotypes accounted for the majority of the African American fragile X population. Third, a putative "protective" haplotype was not found among African Americans, whereas it was found among whites. Fourth, in contrast to that seen in whites, the SNP ATL1 was in linkage equilibrium among African Americans, and it did not add new information to the STR haplotypes. These data indicate that the STR- and SNP-based haplotype associations identified in whites probably reflect the mutational history of the expansion, rather than a mutational mechanism or pathway.

Sp1 binding is critical for promoter assembly and activation of the MCP-1 gene by tumor necrosis factor.

The monocyte chemoattractant protein-1 gene (MCP-1) is induced by the inflammatory cytokine tumor necrosis factor through the coordinate assembly of an NF-kappaB-dependent distal regulatory region and a proximal region that has been suggested to bind Sp1 as well as other factors. To provide a genetic correlation for Sp1 activity in this system, a cell line homozygous for a targeted truncation of the Sp1 gene was derived and examined. We found that the lack of Sp1 binding activity resulted in the inability of both the distal and proximal regions to assemble in vivo even though the binding of NF-kappaB to distal region DNA was unaffected in vitro. We also found that Sp1 and NF-kappaB were the minimal mammalian transcription factors required for efficient activity when transfected into Drosophila Schneider cells. Additionally, Sp3 was able to compensate for Sp1 in the Drosophila tissue cell system but not in the Sp1(-/-) cell line suggesting that Sp1 usage is site-specific and is likely to depend on the context of the binding site. Together, these data provide genetic and biochemical proof for Sp1 in regulating the MCP-1 gene.

Identification of mouse YB1/p50 as a component of the FMRP-associated mRNP particle.

Fragile X mental retardation is caused by the absence of FMRP, an RNA-binding protein found in a large mRNP complex. Although there is evidence that FMRP exists as a homo-multimer, additional proteins have been identified that associate with FMRP in the mRNP. The autosomal paralogs of FMRP, FXR1P, and FXR2P, associate with FMRP, as do nucleolin and NUFIP1, all RNA binding proteins. Using cell lines that were stably transfected with Flag-Fmr1, we identified an additional protein that coimmunoprecipitates with FMRP. The approximately 50 kDa protein was identified by mass spectrometry as mouse Y box-binding protein 1 (YB1), which is 97% identical to the core mRNP protein p50, an RNA-binding protein. An anti-p50 antiserum recognized the 50 kDa protein, confirming the identification. The association of the FMRP-mRNP with a Y box protein, the latter commonly found in mRNPs, further suggests the involvement of FMRP in translation modulation.

Fragile X mouse: strain effects of knockout phenotype and evidence suggesting deficient amygdala function.

Fragile X syndrome is an X-linked form of mental retardation resulting from the absence of expression of the fragile X mental retardation 1 gene. The encoded protein is a ribosome-associated, RNA-binding protein thought to play a role in translational regulation of selective messenger RNA transcripts. A knockout mouse has been described that exhibits subtle deficits in spatial learning but normal early-phase long-term potentiation. We expanded these studies by examination of late-phase hippocampal long-term potentiation, the protein synthesis-dependent form of long-term potentiation, in the Fmrl knockout mice. Here, late-phase long-term potentiation was normal, suggesting either that absence of fragile X mental retardation protein has no influence on long-term potentiation or that any influence is too subtle to be detected by this technique. Alternatively, the hippocampus may not be the primary site affected by the absence of this protein. Accordingly, we examined spatial learning in the knockout mice using the hippocampus-dependent Morris water maze. Contrary to earlier reports, near-normal performance was observed. Since the knockout line used in this study has been back-crossed to C57BL/6 for more than 15 generations, whereas the line used in the earlier studies contained a substantial strain 129 contribution, we examined F1 siblings of knockout and 129 crosses. Here, significant but subtle increased swim latencies in reversal trials were observed, in agreement with the previous studies. These data suggest strain differences between C57BL/6 and 129 that influence the Fmrl knockout phenotype. In order to investigate a paradigm less dependent on hippocampal function, the knockout mice were examined using the conditional fear paradigm. Here, the knockout animals displayed significantly less freezing behavior than their wild-type littermates following both contextual and conditional fear stimuli. These data suggest that amygdala disturbances may also be involved in fragile X syndrome.