The Pathophysiology of Fragile X Syndrome - Emory University

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Annu. Rev. Genom. Human Genet. 2007.8:109-129. Downloaded from arjournals.annualreviews.org by EMORY UNIVERSITY on 08/31/09. For personal use only.

The Pathophysiology of Fragile X Syndrome Olga Penagarikano,1 Jennifer G. Mulle,1 and Stephen T. Warren1,2,3 1

Department of Human Genetics, 2 Department of Biochemistry and 3 Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322; email: [email protected], [email protected], [email protected]

Annu. Rev. Genomics Hum. Genet. 2007. 8:109–29

K ey Words

First published online as a Review in Advance on May 3, 2007.

mental retardation, trinucleotide repeat expansion, FMR1, FMRP, synaptic plasticity, autism

The Annual Review of Genomics and Human Genetics is online at genom.annualreviews.org This article’s doi: 10.1146/annurev.genom.8.080706.092249 Copyright c 2007 by Annual Reviews. All rights reserved 1527-8204/07/0922-0109$20.00

A bstract Fragile X syndrome is the most common form of inherited mental retardation. The disorder is mainly caused by the expansion of the trinucleotide sequence CGG located in the 5 UTR of the FMR1 gene on the X chromosome. The abnormal expansion of this triplet leads to hypermethylation and consequent silencing of the FMR1 gene. Thus, the absence of the encoded protein (FMRP) is the basis for the phenotype. FMRP is a selective RNA-binding protein that associates with polyribosomes and acts as a negative regulator of translation. FMRP appears to play an important role in synaptic plasticity by regulating the synthesis of proteins encoded by certain mRNAs localized in the dendrite. An advancing understanding of the pathophysiology of this disorder has led to promising strategies for pharmacologic interventions.

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IN TRODUC TIO N F X : fragile X


F ragile sites: chromosomal regions that appear as nonrandom chromatin discontinuities at metaphase during cell division F X S: fragile X syndrome

In 1911, Thomas H. Morgan first pointed out sex-linked inheritance of genetic traits in Drosophila melanogaster when he noted that the phenotype “white-eyed” affected only males. Morgan suggested that there could be a recessive genetic trait affecting only males if the gene responsible for it was located on the X chromosome, in the region not common with the Y chromosome. Twenty-five years after Morgan described sex-linked inheritance in flies, human studies were noting a higher incidence of mental retardation in males compared with females (90), and the first pedigrees showing mental retardation associated to the X chromosome were published (79). However, the idea of human intelligence being affected by X-linked genes was not fully appreciated until 1974, when Lehrke published that 25–50% of mental retardation may be attributed to X-chromosome mutations (73). Although this estimation was later discovered to be too high, his work established the concept of X-linked mental retardation (XLMR). Today, approximately 2% of the general population has an intelligence quotient (IQ) below what is considered normal (IQ < 70) and 15–

20% of this is believed to be due to XLMR, with 202 conditions described, of which, to date, 45 have the responsible gene cloned (22). Although each of these X-linked MR loci contributes a relatively small percentage of human XLMR, one locus, that responsible for fragile X syndrome, contributes the largest fraction.

F R A G I L E X SY N D R O M E One of the first large families where mental retardation was transmitted in an X-linked fashion was described by Martin & Bell in 1943 (79). In 1969, Lubs detected an unusual secondary constriction at the end of the long arm of the X chromosome in four mentally retarded males and three obligate carrier females in a single family (76) (F igure 1). However, this observation remained a mystery for more than a decade until Sutherland showed that specific culture conditions are needed for the cytogenetic expression of the “marker X,” enabling the recognition of additional families (105). Fragile sites are named with the term FRA, designating fragility, followed by the chromosome where they are located and a letter indicating the order in which they were found (14). Thus, the fragile site associated to the fragile X chromosome, now localized specifically to band Xq27.3 (53), was named FRAXA, as it was the first fragile site described on chromosome X. Richards & Sutherland (93) later analyzed the original pedigree described by Martin and Bell and showed that the affected males did indeed have fragile X syndrome. Consequently, the descriptors Martin-Bell syndrome or marker X syndrome have now been largely superseded by the designation fragile X syndrome (FXS).

F RA G I L E X P H E N O T YP E F igure 1 The “marker X” chromosome. Metaphase chromosomes showing the peculiar constriction at the end of the long arm of chromosome X that is characteristic in fragile X (FX) individuals. 110

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Initially, the fragile or marker X chromosome was not clearly associated with any other phenotype but mental retardation. However, once the laboratory conditions necessary for the reproducible expression of the cytogenetic


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fragile site were established, a number of affected families were detected and the associated phenotype emerged. Mental retardation is the most prominent phenotype, with IQ values typically between 20 and 70 (41). The cognitive dysfunction particularly affects short-term memory for complex information, visuospatial skills, and speech. A delay in speech is common and often the first symptom that brings the child to medical attention. Some patients show hyperactivity, hypersensitivity to sensorial stimuli, and attention deficit, and between 15–50% of affected individuals show some autistic behavior such as poor visual contact, tactile defensiveness, and repetitive behaviors (26). Individuals affected by FXS show a subtle, yet characteristic, facial morphology of macrocephaly, with a long narrow face and prominent forehead, jaw, and ears (23) (F igure 2). Macroorchidism, or enlarged testicles, are commonly seen in postpubescent male patients. Other physical features such as the presence of a high arched palate, hypotonia (51), flat feet, increased joint laxity, and mitral valve prolapse (74) have also been reported. These findings are collectively believed to be due to a poorly understood connective tissue abnormality that has received little research attention (58). Several investigators have also reported a neuroendocrine dysfunction in FXS. Although poorly understood at the molecular level, an unusual overgrowth pattern has been attributed to this and includes an increased birth weight, macrocephaly, and increased stature, particularly in childhood. Some individuals affected by FXS have also been diagnosed as Sotos (115) or Prader-Willi syndromes (32) due to their striking physical similarity. Endocrine studies of these patients have reported hypothalamus dysfunction (55).

U N USU A L I N H E RI T A N C E PA T T E R N As soon as families showing the FX chromosome were identified, it became apparent

F igure 2 The fragile X phenotype. Note the characteristic facial features of the syndrome: long, narrow face, prominent forehead, jaw, and ears.

that the inheritance pattern was not consistent with a recessive X-linked condition, as previously thought. The main conflicting data were the existence of nonaffected male carriers and affected female carriers (84). Sherman et al. (99, 100) conducted extensive pedigree analyses and reported that 20% of males carrying the mutated gene were not affected. These individuals were termed normal transmitting males (NTMs). In addition, 30% of carrier females showed some form of mental www.annualreviews.org • Fragile X Syndrome

M acroorchidism: testicular volume >30 ml, which is principally developed during the postpubertal period N T M : normal transmitting male

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F M R1: fragile X mental retardation 1


M icrosatellite: repetitive DNA sequence consisting of 1–4 base repeat units D ynamic m utation: A repetitive DNA sequence whose increasing length, beyond a certain threshold, confers an increasing inter-generational instability

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impairment. They also noticed that the risk of inheriting the syndrome depended on the position of an individual within the pedigree: The mothers of NTM showed three times less risk of having affected sons than their daughters. This phenomenon is known as the Sherman paradox and was not resolved until 1991, when the gene responsible for the syndrome was identified and, at the same time, a new mutational mechanism revealing the particular inheritance model: trinucleotide repeat expansion.

A N E W C O N C EP T O F M U TAT IO N According to Mendel’s work in 1860, the inheritance of genetic traits is determined by units of discrete nature, later named genes, which are transmitted intact and independently to the next generation. Almost 100 years later Benzer showed that genes could suffer mutations, but, once produced, these mutations would be transmitted intact to the next generation. This concept is still valid for most genes; however, since 1991, when the gene responsible for FXS was cloned, it has been revealed that specific repetitive sequences in some genes are unstable and can mutate serially upon transmission.

T he F ragile X M utation The existence of the Xq27.3 cytogenetic fragile site associated with the syndrome was a target for subsequent molecular studies. Pedigree analysis localized both the causal locus and the fragile site to a 22-cM region on the X chromosome (88), and further studies revealed a number of linked markers that reduced the interval to 1–2 Mb and strengthened the localization of the disease locus to the fragile site (57, 95, 106, 107). Warren et al. (117) described somatic cell hybrids carrying the human fragile X chromosome with rodent chromosome translocations at the FX site. Heitz et al. (54) identified a yeast artificial chromosome (YAC) containing two 112

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markers known to flank the fragile site hybrid break points and showed that this YAC contains a CpG island aberrantly methylated in fragile X patients. Furthermore, these abnormally methylated fragments were unstable in affected individuals and increased in size when transmitted through a pedigree (88). In 1991, Verkerk et al. (114) used these somatic cell hybrids and identified a gene, expressed in the brain, that showed a tandem repeat of the CGG trinucleotide in its 5 region, which was expanded in affected individuals and was coincident with the fragile site. The gene was named Fragile X Mental Retardation 1 (FMR1), assuming that it was the first of a number of yet-identified genes associated with the FX site. We now appreciate that the FMR1 gene resides in a gene desert, and this single gene is responsible for the syndrome. Microsatellite sequences, such as the one responsible for FXS, are very frequent in the human genome, being present every 2 kb and representing approximately 90 Mb of the human genome (62). Most of them show relatively high levels of polymorphism, but are usually stable upon transmission. The variability in the repeat length represents a slight instability in the sequence that usually appears as an expansion or contraction of one or two repeats (12). These small changes in repeat length usually remain in the “normal” range; therefore, most microsatellites are not associated with any disease. However, trinucleotide repeats can show a much greater instability. In particular, when the transmitting allele is beyond a certain threshold size, it generates a new lengthened allele when transmitted that is often a large expansion that leads to genetic disorders. This new and unexpected multistep mutational mechanism is called dynamic mutation (94). FXS, along with Kennedy disease [spinal-bulbar muscular atrophy (SBMA)], was the first to be identified among the disorders associated with trinucleotide repeat expansion. To date, at least 16 disorders are known to be caused by trinucleotide repeat expansion (28). The trinucleotide repeat expansion can lie within the coding region


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of the gene, as in Kennedy disease or other polyglutamine diseases, where the expanded CAG triplet, which codes for glutamine, is included in the encoded protein and the new mutant protein gains a novel deleterious function. Alternatively, the triplet expansion can be located in a noncoding region, as in FXS, and, depending on the triplet and on the location (within an intron or within a nontranslated region of an exon), the pathogenic mechanism is different, usually leading to a diminished expression or even silencing of the gene.

occur; massive expansions could take place by some continuous slippage due to a block in the replication process generated by alternative structures formed by the trinucleotide repeat sequences (89). Because slipped-strand mispairing during DNA replication is the most accepted mechanism of trinucleotide repeat expansion, it is natural to think that the mismatch repair system (MRS) would play an important role. However, to date no clear data exist regarding its role in triplet repeat instability.

T rinucleotide R epeat E xpansion

T H E FMR1 G E N E

Since the molecular basis of this new mutational mechanism was described, special efforts have been made to explain the phenomenon of expansion. The proposed models include unequal exchange of genetic material during meiosis or mitosis due to an incorrect pairing of the chromatids along the repetitive sequence and gene conversion. Although these mechanisms could be the cause of some of the expansions observed in trinucleotide repeats, the initial event leading to the instability of a normal allele thought to be slipped-strand mispairing during DNA replication (70). Richards & Sutherland (93) suggested that the slippage would occur specifically in the Okazaki fragments. Okazaki fragments formed by long or uninterrupted repetitive sequences will not be anchored by unique sequences at their ends, showing a risk of suffering slippage. If slippage occurs, DNA repairing enzymes could add nucleotides to the template strand to make it complementary to the newly formed strand, generating an expansion. In support of this, the absence of a functional flap endonuclease 1 (FEN-1), which is necessary for correct processing of Okazaki fragments, has dramatic effects on repeat instability. Indeed, FEN-1 activity is diminished when the flap length is longer than 11 repeats (72). If the flap is not correctly processed, it will lead to an expansion of the repeat. Initially, a gradual accumulation of one or a few repeats per generation would

The FMR1 gene is 38 kb in length and contains 17 exons (F igure 3). The 4.4-kb fulllength mRNA codes for a protein with a maximum length of 632 amino acids and a molecular mass of 80 kDa, although different transcripts can be produced by alternative splicing (7). The translation start is located 69 base pairs (bp) downstream the repetitive region and the repeat lays in the 5 untranslated region (UTR) within the first exon of the gene (7). In addition, the gene shows a CpG island located 250 bp upstream from the repeat (38). The repeat length is polymorphic and ranges from 6–54 CGG in normal individuals (44). Female carriers and NTMs show an expansion between 55 and 200 repeats, termed premutation. When the expansion is more than 200 repeats, it is termed full mutation. As a result of the expansion, the repeat, the upstream CpG island, and the surrounding sequence become hypermethylated and the gene silenced. Thus, the absence of the encoded protein is the basis for the phenotype (91). Silencing the expanded FMR1 allele is a unique example of a local epigenetic change driven by variation in the gene sequence. Methylation of the FMR1 gene acts both directly, by inhibiting the binding of transcription factors, and indirectly, by inducing chromatin condensation, which, in turn, prevents the transcription machinery from binding. The transcriptionally inactive status of the FMR1 gene shows four occupied www.annualreviews.org • Fragile X Syndrome

O kazaki fragment: short fragment of DNA created on the lagging strand during DNA replication due to the intrinsic 5 –3 polarity of DNA polymerase F lap endonuclease 1 ( F E N -1): removes 5 flaps from Okazaki fragments. Flaps containing repeats could acquire different DNA conformations that enable them to escape FEN-1 cleavage

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Transcription start ATG

TAA Introns

–129

–94

– 66

– 44

+1

Splicing

+130

GC rich GC boxes E-box-CRE palindrome

1

2 3 4 5

(CGG)n Normal: 6-44 Intermediate: 45-54 Premutation: 55-200 Full mutation: >200


Exons

6 7

NLS

8

9

KH1

10 11 KH2

1213 14

15 16

17

NES RGG

F igure 3 Schematic representation of the fragile X mental retardation 1 (FMR1) gene. The FMR1 gene shows 17 exons that undergo alternative splicing. The CGG repeat is located within the untranslated region of the first exon. Expansion over 200 repeats leads to methylation of the promoter and transcriptional silencing. The location of the principal domains of the normally encoded protein is also indicated.

transcription factor-binding sites in its promoter, including a palindrome, two GClike boxes, and an overlapping E-box-cAMP response element (CRE) site. Studies on the proteins that bind to these sites have identified the factors upstream stimulatory factor 1 (USF1) and USF2, nuclear respiratory factor 1 (NRF1) and NRF2, specificity protein 1 (Sp1) and cAMP response element binding protein (CREB) as the most actively involved in FMR1 transcriptional activity (69, 103). In addition, the 5 region of the FMR1 gene is normally associated with histone proteins H3 and H4 acetylated in their lysine residues, but this acetylation is reduced in FX cells (25). Changes in histone methylation have also been described, with histone 3 showing methylated lysine 4 and unmethylated lysine 9 in normal cells but the opposite methylation pattern in FX cells (24). Attempts to pharmacologically reactivate the gene have shown a synergistic effect using both DNA methylation and histone deacetylation inhibitors (20); however, the connection between histone modifications and DNA methylation remains unclear. It was recently shown that the transcriptionally active versus inactive state of the FMR1 gene display different broader chromatin conformations. Chromatin segments located throughout a 50-kb domain around the promoter interact less fre114

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quently when FMR1 is expressed compared with when the gene is repressed. Therefore, the expression-correlated change in conformation affects a significantly larger domain than the one marked by histone modifications (46). However, the cause-and-effect relationship among promoter activation, local histone modifications, and broader changes in chromatin remains to be determined. All data suggest that the transcriptional repression of the FMR1 gene occurs when both the CpG island and the CGG repeat become hypermethylated. It is assumed that methylation and consequent inactivation of the gene occur after fertilization during embryogenesis and that the protein is expressed during the first stages of embryonic development (75, 119). However, this early expression does not seem to play an important biological role as individuals affected as consequence of deletions in the FMR1 gene, and therefore not expressing the protein at any point during their development, do not show any phenotypic differences from individuals affected owing to gene methylation (45, 120).

F actors I n fl uencing C G G R epeat E xpansion With FXS, as in every dynamic mutation, there is a multistep process of expansion that


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takes place over many generations. Normal repeats (6–54 CGG) are usually transmitted stably, but sometimes increase (or decrease) by a few repeats, creating a pool of at-risk alleles that can eventually become premutations. Premutations (55–200 CGG) tend to expand when maternally transmitted, as male spermatogenesis is unable to maintain full-mutation alleles (78). The probability of expansion depends on the repeat length. Alleles larger than 90–100 CGG repeats show an almost 100% risk of expansion to full mutation (>200 CGG) in the next generation (86). Smaller alleles, although unstable, usually do not expand to a full mutation in one generation. To date, the smallest allele that has suffered that kind of change has 59 repeats (87). In addition, sequencing analysis of normal FMR1 alleles revealed that the CGG repeat is not pure, but presents an AGG interruption every 9–10 repeat units, and the most common allele is (CGG)9 − 10 AGG(CGG)9 AGG(CGG)9 (71). However, in FX families the premutated alleles usually have one or no interruptions. Loss or lack of one of these interruptions is an important determinant for repeat instability (37). The 59 repeat allele that expanded to a full mutation in one generation had no interruptions, supporting their important role. The fact that most of the premutation alleles bigger than 90 repeats expand to full mutation in one generation, and assuming that the maximum number of AGG interruptions that they show is probably 2, and that they are located every 9–10 CGG, suggests that approximately 70 pure repeats are needed for the allele to be fully expanded. Because the most likely mechanism of expansion is slipped-strand mispairing during DNA replication in the Okazaki fragments, which range among 25–300 nucleotides, and 70 repeats make 210 nucleotides, there is a high probability that this fragment is not anchored by unique flanking sequences, which supports the idea of strand slippage. Analysis of polymorphic markers within and near the FMR1 gene shows that certain

haplotypes are preferably associated with the FX mutation. This observation is denoted as linkage disequilibrium and suggests the existence of a pool of founder chromosomes that lead to the FX chromosomes (71). Thus, apparently nonrelated individuals could have common ancestors, and the origin of the mutation could be the same. Different mutational routes that correspond to the different haplotypes observed in FX individuals may exist. They typically involve the generation of alleles with longer tracts of pure CGG repeats either by loss of AGG interruptions or by gradual increase in the repeat length. It is still not clear when the repeat expansion occurs. First, the observation that affected individuals are mosaics, showing both premutation and full-mutation alleles or fullmutation alleles of different sizes, together with the fact that affected males show only premutations in their sperm (42) led to the assumption of a postzygotic model, where the zygote harbors a premutation and the expansion occurs mitotically during early embriogenesis. It was later pointed out that there was no correlation between the repeat length and the degree of mosaicism and that no mechanism that distinguishes a maternal transmission, suffering expansion, from a paternal transmission, not suffering expansion, was known. Therefore, the mosaicism observed in patients would likely be due to the contraction of somatic alleles rather than to the different expansion of maternal premutated alleles during gametogenesis, and expansion would probably be prezygotic (83). The prezygotic model of expansion is supported by the observation of full-mutation alleles in oocytes and in fetal spermatogonia, but only premutations in testes at a later stage, suggesting that the expansion must occur before germinal differentiation (78). Animal models will be essential to determine the timing and the mechanism of both the expansion and the methylation and inactivation seen in humans. However, although some expansions were recently reported in a mouse containing a human (CGG)98 repeat www.annualreviews.org • Fragile X Syndrome

L in kage disequilibriu m: nonrandom association of alleles at two or more linked loci

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P O F : premature ovarian failure F X T AS: fragile X tremor ataxia syndrome

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(15) (even occasionally reaching the fullmutation range), to date there are no reports of massive expansions occurring in the mouse at the same frequencies as are observed in human pedigrees. In addition, mice with long repeats ( 230 CGG) show an absence of abnormal methylation, indicating that the FX full mutation has not yet been mimicked in mice.


F ragile X Syndrome W ithout C G G E xpansion Because the cause of the phenotype associated with FXS is the lack of the protein encoded by the FMR1 gene, it is reasonable to think that other mutations apart from the CGG expansion could lead to the disease. Actually, more than 15 deletions affecting part of or the entire gene, and ranging from 1.6 kb to 13 Mb, have been described (45, 52, 56, 63, 81, 109, 120). Besides deletions, point mutations that lead to the phenotype due to the production of a nonfunctional protein have also been described (31, 77). It is important to consider that most diagnostic testing for FXS is limited to the examination of the CGG repeat and largely ignores conventional mutations. However, because the full mutation leads to a functional null allele, it is likely that there are many nonsense and missense mutations of FMR1 that are being missed.

F RA G I L E X PR E M U T AT I O N For many years, male and female premutation carriers were considered asymptomatic. This view was gradually challenged by reports of different premutation-associated clinical phenotypes over the past decade. The prevalence of premutation alleles >54 repeats in the general population is estimated at 1/259 females and 1/813 males (96). As early as 1986, Fryns (43) noted an increased rate of twinning among female premutation carriers, suggesting the possibility of ovarian dysfunction. A few years later, Cronister et al. (27) noticed that women carrying the FX mutation who 116

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were cognitively normal had a higher prevalence of premature ovarian failure. The mean age of menopause in the general population is 51. It is assumed that it occurs as a result of the reduction of the initial oocyte pool below a determined level. Premature ovarian failure (POF) is defined as menopause before 40 years of age. Both genetic and environmental causes are known (97), but 60% of the cases have no obvious cause. When premutation carriers could be distinguished from mutation carriers, it was shown that only the former showed this higher prevalence (16–24%), whereas the latter showed the same risk as noncarrier relatives and the general population ( 1%) (3). In addition, an increased level of follicle-stimulating hormone (FSH), which is as an indicator of ovarian aging, was found in premutation carriers (104). Because only a proportion of women who carry the premutation have ovarian dysfunction, it is important to identify the risk factors that lead to clinical variability. The most obvious factors are those related to the FMR1 gene. In a recent report, Sullivan et al. (104) found a significant influence of repeat size on the risk for ovarian failure, with increasing prevalence of POF and decreasing age at menopause correlating with an increasing repeat size up to 100 repeats. More recently, an emerging group of premutation carriers showing neurological problems was identified. Hagerman et al. (50) identified five male patients with clinical features of intention tremor and cerebellar ataxia at 50–60 years of age. The disorder was designated as fragile X tremor ataxia syndrome (FXTAS). Prior to onset, these patients had normal cognitive ability. It was later shown that this phenotype is not exclusive to males, as earlier suggested, but that females can also be affected, although they show a later onset, likely due to the degree of inactivation of the affected X chromosome (49). These patients show a general reduction in brain size together with cerebellar damage, severe loss of Purkinje cells, spongiosis of the white matter, and axon dystrophy. In addition, eosinophilic intranuclear inclusions,


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mainly formed by RNA and proteins, in neurons and astrocytes throughout the brain were found in postmortem samples obtained from symptomatic male premutation carriers (47). These types of granules have also been found in other neurodegenerative diseases caused by trinuclotide repeat expansion, such as polyglutamine diseases and myotonic dystrophy. The existence of phenotypes associated exclusively with the FX premutation suggests that a molecular cause other than the reduction in FMRP levels must exist. mRNA levels increased up to tenfold in premutation carriers, despite the reduction of FMRP expression (110). Transcriptional activity seems to increase in response to a decreased translation rate (40). A dominant RNA gain-of-function mechanism has been proposed as the molecular mechanism underlying FXTAS. The elevated number of FMR1 transcripts present in premutation carriers might result in sequestration of cellular proteins and formation of inclusions. A mouse model for FXTAS that displays similar neuronal intranuclear inclusions was recently generated (112). Further research on the development and composition of the inclusions will help scientists to better understand the mechanism of RNAmediated neurodegeneration. Ovarian dysfunction could also result from a toxic effect on the follicles of an increased amount of FMR1 RNA, leading to increased follicular atresia rates rather than a reduction of the initial oocyte pool size. Future studies on ovarian tissue from premutation carriers will be necessary to investigate this hypothesis.

F ragile X M ental R etardation P rotein Understanding the pathogenesis of FXS involves studying its cause and, thus, the function of the molecule absent in affected individuals: FMRP. The protein encoded by the FMR1 gene is expressed widely, particularly in the brain and testes (33), which is consistent with the primary characteristics of the syndrome, mental retardation, and

macroorchidism. As a result of alternative splicing, the FMR1 gene can generate at least 12 different proteins between 67–80 kDa (33, 101). Their expression does not seem to be tissue specific, as different tissues show the same expression pattern (113). Alternative splicing affects exons 12, 14, 15, and 17. In general, the most common isoforms lack exon 12, and isoforms lacking exon 14 show the lowest expression (102). FMRP is predominantly localized in the cytoplasm; however, it was shown that isoforms lacking exon 14 were localized to the nucleus, consistent with the identification of a nuclear exportation signal (NES) encoded by this exon. FMRP also contains a nuclear localization signal (NLS) in exons 1– 5, indicating that FMRP shuttles between the nucleus and cytoplasm. This has been corroborated by electron microscopy (39). FMRP shows sequence motifs characteristic of RNA-binding proteins. It has two heterogeneus nuclear ribonucleoprotein K Homology (KH) domains in exons 8 and 10, an Arginine-Glycine-Glycine (RGG) box in exon 15 (7), and an RNA-binding domain in the amino-terminal region of the protein (2). In vitro it binds to approximately 4% of the mRNAs in fetal human brain, including its own message (7). FMRP has also been shown to associate with polyribosomes in an RNAdependent manner via messenger ribonucleoprotein particles (mRNP) of about 660 kDa, which contain several other proteins including its two autosomal homologs fragile X related protein 1 (FXR1P) and FXR2P encoded by autosomal genes localized to the chromosomal regions 3q28 and 17p13.1, respectively (123). FXR1P and FXR2P show a 86% and 70% level of similarity with FMRP, respectively, and show the same principal functional domains as FMRP: NLS and NES, the KH domains, the RGG box, and a homodimerization domain. They are mainly localized to the cytoplasm associated with polysomes as part of the same protein complex. They also shuttle between the nucleus and cytoplasm, and FXR2P shows a nucleolar localization signal (NoS) and shuttles between the www.annualreviews.org • Fragile X Syndrome

F M R P: fragile X mental retardation protein

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Synaptic plasticity: ability of the synapse to change in strength or change in transmembrane potential

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cytoplasm and the nucleolus (108). In addition to FMRP homologs, other proteins that interact with FMRP or that are present in the same complex have been identified. However, it has not been determined whether all complexes are the same or if there are differences in the associated proteins. If they were different, each complex could be affected by the absence of FMRP in a different way. These proteins include nuclear FMRP interacting protein (NUFIP), cytoplasmic FMRP interacting protein 1 (CYFIP1) and CYFIP2, an 82-kD FMRP interacting protein (82-FIP), nucleolin, and Y-Box factor 1/p50 (YB1/p50) (10, 11, 17, 18, 98). Posttranslational modifications of FMRP have also been reported. FMRP was recently shown to be normally methylated in the RGG box, mainly in arginine 544, and this methylation seems to regulate its protein-protein and protein-RNA interactions (35). FMRP also shows a cluster of phosphorylated residues on the N-terminal side. The primary site of phosphorylation is at serine 499 and it triggers phosphorylation of nearby residues when the residue is phosphorylated. This phosphorylation does not affect the rate of degradation of FMRP or the species of RNA with which FMRP is associated; rather, it has been observed that the phosphorylated form of FMRP is associated with apparently stalled polyribosomes whereas the unphosphorylated form is associated with actively translating polyribosomes, suggesting that phosphorylation could regulate FMRP function (19). Identification of FMRP mRNA ligands in vivo will lead to essential information about the functional roles of FMRP. Several works have focused on the search for mRNAs bound to the protein (16, 20, 29, 82). These experiments led to the identification of mRNAs that encode for proteins with an important role for neuronal function, synaptic plasticity, and neuronal maturation. Efforts are being made to elucidate the way that FMRP regulates its target mRNAs. On one hand, FMRP could recognize its targets directly. Darnell et al. (29) showed that the RGG box Penagarikano

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binds to an RNA loop structure named G quartet, where four guanines are stabilized by Hoogsteen-type hydrogen bonds in a planar conformation. Most of the mRNA targets identified for FMRP in the above-mentioned studies contain a potential G-quartet structure (F igure 4). Chen et al. (20) identified another set of RNA ligands containing U-rich sequences with 5–23 bases of repeating U pentamers. In addition, the KH2 domain has been shown to bind a complex RNA tertiary structure named loop-loop pseudoknot or “kissing complex” (30). The authors showed that RNAs with this structure can compete off the association of FMRP with polyribosomes, suggesting that this structure could be crucial for FMRP translational regulation. On the other hand, an indirect or more complex interaction between FMRP and its target mRNAs could occur. Recent data have shown the association of FMRP with the microRNA pathway, which suggests a new way that FMRP could modulate translation. MicroRNAs are short (21 nucleotides) noncoding regulatory RNAs that modulate gene expression by blocking translation of partially homolog mRNAs. MicroRNAs are processed from longer RNAs by an RNAse III enzyme named Dicer and are loaded into an RNAinduced silencing complex (RISC) protein complex, which mediates the interaction between microRNAs and their mRNA targets. FMRP associates with both microRNAs and proteins from the RISC complex as well as with Dicer activity, suggesting that it could also be involved in processing microRNA precursors (66). The “kissing complex” may represent the interaction between an mRNA and a microRNA or other noncoding RNA. FMRP was reported to bind to the small nonmessenger cytoplasmic RNA BC1, which simultaneously associates to mRNA targets known to be regulated by FMRP (122). BC1 has been suggested to mediate the formation of a translational inhibition complex by association to a polyA-binding protein and eIF4A (116). However, BC1 is found in lighter polysome fractions than FMRP,

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46

a 47 G 48 G

GUC AA

G

34

GG


c AGGGU

A

G

G 38 U A G

U G 31 U G G C G U C G G 5'

AUGU

37

42

33

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making their direct interaction unlikely (68). Recent data support the interaction of FMRP with the RISC complex in regulating synaptic protein synthesis. FMRP interacts with the calcium-calmodulin-dependent kinase II (CaMKII) mRNA, from which dendritic local protein synthesis is required for memory formation (48). Translation of this mRNA is inhibited by RISC and relieved by neuronal stimulation, associating the miRNA pathway with the control of local protein synthesis in neurons (8).

A G A A G A

ACCAGA A U U C CG U G A G GA

AU CAG

A G G

F igure 4 RNA structures recognized by fragile X mental retardation protein (FMRP). (a) G quartet, (b) U pentamer, (c) kissing complex.

N E UR O N AL-M O L E C U LAR CO N NEC TIO N FMRP expression is widely seen during mouse embryogenesis. During development, the expression level diminishes in some tissues, leading to a more specific expression pattern (9). In humans, embryonic studies have shown FMRP expression in the central nervous system of a 9-week-old fetus whereas in a 25-week-old fetus FMRP expression was restricted to neurons (1). Autopsies performed in individuals affected with FXS have www.annualreviews.org • Fragile X Syndrome

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D endritic spine: small membranous extrusion that protrudes from a dendrite (branched projections of a neuron) and forms one half of a synapse Synapse: specialized junction through which cells of the nervous system signal to one another from axons to dendrites L T P: long-term potentiation L T D : long-term depression

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made the associated brain abnormalities apparent. They show more immature (longer and thinner) dendritic spines, where most of the synapses occur, and a higher density of them, suggesting a possible misregulation in their development and elimination (65). An abnormal development of dendritic spines in FMR1 knockout mice has also been reported (85). During development, synapses are often produced in numbers that exceed those that will ultimately survive. Maturation and pruning are therefore important to establish the final synaptic pattern. Abnormalities in the dendritic spines have also been associated with nonsyndromic mental retardation as well as with Down, Rett, CoffinLowry, and Rubinstein-Taybi syndromes (34). One of the neurological problems in FXS likely associated with this spine morphology is the presence of epileptic seizures because the characteristic spine morphology could cause a greater neuronal excitation (64). Specific proteins and biochemical pathways have been associated with spine morphogenesis; many of these pathways ultimately lead to actin rearrangement in the spine cytoskeleton. The local synthesis of FMRP can potentially influence other proteins or signaling cascades involved in morphogenesis such as Rac1, MAP1B, CaMKII, calbindin, alpha-glucocorticoid receptor, and cadherins (48). Thus, FXS is considered a synaptic disease. FMRP is an RNA-binding protein associated with polyribosomes, suggesting that it has a role in modulating protein synthesis in neurons, where it is largely expressed. In neurons, protein synthesis in both cell bodies and dendritic spines is important for synaptic plasticity, and certain mRNAs are translated in response to a specific synaptic activation. Synaptic plasticity is essential for memory and learning processes and involves long-term potentiation (LTP) and long-term depression (LTD), which are associated with synapse creation and elimination in addition to synaptic transmission. Depending on the brain region, synaptic plasPenagarikano

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ticity is expressed pre- and/or postsynaptically. At the postsynaptic level, LTD can be triggered by the activation of postsynaptic N-methyl-D-aspartate (NMDA) receptors or group I metabotropic glutamate receptors (mGluRs). Both types of LTD do not occlude each other, but result in a decrease of surface expression of alpha-amino-3hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (61). The activation of group I mGluRs (mGluR1 and mGluR5) was reported to stimulate the synthesis of FMRP in synapses (118) and in neuronal lysate (59). Fmr1 knockout mice (36) showed enhanced mGluR-LTD in the hippocampal Schaffer collateral synapses of the CA1 area (60) and in the cerebellar parallel fiber to Purkinje cell synapses (67). NMDAR-LTD in Fmr1 knockout mice remained the same as that in the wild-type control, indicating that the phenotype was specific to the mGluR-dependent form of synaptic plasticity. In addition, protein synthesis dependent on mGluR activation was enhanced in the absence of FMRP (60). These data led to the hypothesis that FMRP normally represses further protein synthesis (13). It was recently shown that the rapid increase in FMRP synthesis associated with the stimulation that can cause mGluR-LTD is transient and followed by a rapid decrease in FMRP back to baseline levels, suggesting that FMRP is dynamically regulated during mGluR-LTD, with rapid synthesis followed by rapid degradation in hippocampal slices and homogenate. The authors also showed that the ubiquitin-proteasome degradation pathway played a role in FMRP regulation after mGluR stimulation because FMRP was polyubiquitinated in the same temporal window where its levels begin to decline (59). Therefore, it is reasonable to propose that the rapid degradation of FMRP might be required to permit its target mRNAs to be translated and consolidate mGluR-LTD. In addition to local protein synthesis at synapses, FMRP appears to be involved in the mRNA transport to the synaptic locations. Stimulation of mGluRs increases the transport of

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FMRP-containing granules to the dendrites (5, 6). Together, these data suggest a dual role for FMRP: delivery and release of mRNAs for activity-dependent translation.


T REAT M E N T Useful guidelines for health supervision of FX children were published by the American Academy of Pediatrics (4) and include advice for both physical and behavioral components of the syndrome. The therapy currently used in FX individuals is designed specifically for each patient and is focused on his/her specific behavior symptoms. Unfortunately, there is not yet a treatment to compensate for the absence of FMRP. Attempts have been made to reactivate the silenced FMR1 gene and therefore restore the production of the protein (21, 92); however, reactivation processes are too general and genes other than FMR1 may be reactivated. In addition, toxicity of such approaches is very high. Regardless, reactivation of a full mutation would still produce a transcript with a long repeat that renders the message inefficiently translated. The possibility of gene therapy is currently not possible due to difficulties in the delivery of the normal gene into neurons and its restoration of the phenotype. More recently, the understanding of the function of FMRP and the consequence of its absence has led to potential therapeutic approaches. Because FMRP is a translational suppressor and mGluR 5 activation of the excitatory pathway stimulates local protein synthesis, the absence of FMRP leads to the apparent overstimulation of translation by mGluR5 signaling (13). This imbalance, which has fundamental consequences with regard to synaptic plasticity, may be countered by mGluR5 antagonists (F igure 5). One such antagonist is MPEP. Remarkably, MPEP was recently shown to be highly efficient in rescuing phenotypes associated with FMRP loss in animal models for FXS. In the Drosophila model, McBride et al. (80) showed that MPEP can rescue memory deficits in courtship be-

mGluR1/5

mGluR1/5

Drug targets?

Translation stimulation

mRNA translation

FMRP Normal

mRNA translation

Translation suppression

FMRP Fragile X syndrome

F igure 5 Role of fragile X mental retardation protein (FMRP) in modulating synaptic plasticity. The activation of group I metabotropic receptors (mGluR) stimulates translation of specific mRNAs at synapses. FMRP normally acts as a translational repressor regulating such expression; however, in the absence of FMRP, overexpression of such messages is found.

havior and mushroom body defects observed in mutant flies. Similarly, Yan et al. (121) showed that FMR1 knockout mice treated with MPEP exhibit a reduction in the sensitivity to audiogenic seizures and respond better to the open field test, with a reduced tendency to mainly be in the center of the field. Using zebrafish as a model for the study of human FXS, Tucker et al. (111) recently reported that MPEP was able to rescue the axonal branching defect observed in these animals. These data indicate that the interaction between mGluR signaling and FMR1 function is responsible for some of the symptoms associated with FXS. Thus, drugs targeting mGLuR5 or downstream signaling may be of therapeutic potential in FXS. It is anticipated that clinical trials will begin within the next two years. FXS is instructive regarding genetic disease in general. Once given the “toe hold” of the identification of the responsible gene, basic scientific investigation into the normal and abnormal function of encoded proteins can lead to sufficient molecular understanding to suggest traditional pharmacologic interventions for even the most seemingly intractable disorders. www.annualreviews.org • Fragile X Syndrome

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S U M M A RY P O I N T S 1. FXS is the most common form of inherited mental retardation, has striking behavioral overlap with autism, and may be considered the best-understood form of autism. 2. FXS is caused by the functional absence of FMRP encoded by the FMR1 X-linked gene, usually due to silencing of the gene caused by expansion of the trinucleotide repeat CGG located in its 5 UTR.


3. FMRP is a selective RNA-binding protein associated with polyribosomes and expressed in neurons including the dendritic spine, where it has been shown to regulate translation of mRNAs important for synaptic plasticity and neuronal maturation. 4. How FMRP plays its role in translational regulation is still unclear and involves direct interaction with mRNAs as well as interaction with the microRNA pathway.

F U T U R E ISS U E S T O B E R E S O L V E D 1. Animal models for both expansion of the CGG repeat and silencing of the FMR1 gene will be essential to determine the timing and mechanism of repeat expansion and subsequent epigenetic changes in FMR1. 2. The molecular details of how FMRP regulates mRNA translation need to be elucidated as does the regulation of FMRP itself. 3. A better understanding is needed of the role of FMRP in local protein synthesis in the dendrite, its normal role in synaptic plasticity, and the neuronal consequence of its absence. 4. Pharmacological approaches have been identified that may compensate for the loss of FMRP, and therapeutic approaches now need to be developed to improve the quality of life of patients with fragile X syndrome.

D IS C L O S U R E S T A T E M E N T SW serves as Chair of the Scientific Advisory Committee for Seaside Therapeutics, Inc.

LITERATURE CITED 1. Abitbol M, Menini C, Delezoide AL, Rhyner T, Vekemans M, Mallet J. 1993. Nucleus basalis magnocellularis and hippocampus are the major sites of FMR-1 expression in the human fetal brain. Nat. Genet. 4(2):147–55 2. Adinolfi S, Ramos A, Martin SR, Dal Piaz F, Pucci P, et al. 2003. The N-terminus of the Fragile X mental retardation protein contains a novel domain involved in dimerization and RNA binding. Biochemistry 42(35):10437–44 3. Allingham-Hawkins DJ, Babul-Hirji R, Chitayat D, Holden JJ, Yang KT, et al. 1999. Fragile X premutation is a significant risk factor for premature ovarian failure: the International Collaborative POF in Fragile X study—preliminary data. Am. J. Med. Genet. 83(4):322–25 122

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4. American Academy of Pediatrics Committee on Genetics. 1996. Health supervision for children with Fragile X syndrome Pediatrics. 98(3 Pt 1):467–72 5. Antar LN, Dictenberg JB, Plociniak M, Afroz R, Bassell GJ. 2005. Localization of FMRP associated mRNA granules and requirement of microtubules for activity-dependent trafficking in hippocampal neurons. Genes Brain Behav. 4:350–59 6. Antar LN, Afroz R, Dictenberg JB, Carroll RC, Bassell GJ. 2004. Metabotropic glutamate receptor activation regulates Fragile X mental retardation protein and FMR1 mRNA localization differentially in dendrites and at synapses. J. Neurosci. 24(11):2648–55 7. Ashley C T J, W ilkinson K D , R eines D , Warren S T . 1993. F M R1 protein: conserved R N P family domains and selective R N A binding. Science 262(5133):563–66 8. Ashraf SI, McLoon AL, Sclarsic SM, Kunes S. 2006. Synaptic protein synthesis associated with memory is regulated by the RISC pathway in Drosophila. Cell 124:191–205 9. Bakker CE, de Diego Otero Y, Bontekoe C, Raghoe P, Luteijn T, et al. 2000. Immunocytochemical and biochemical characterization of FMRP, FXR1P, and FXR2P in the mouse. Exp. Cell. Res. 258(1):162–70 10. Bardoni B, Castets M, Huot ME, Schenck A, Adinolfi S, et al. 2003. 82-FIP, a novel FMRP (fragile X mental retardation protein) interacting protein, shows a cell cycle-dependent intracellular localization. Hum. Mol. Genet. 12(14):1689–98 11. Bardoni B, Schenck A, Mandel JL. 1999. A novel RNA-binding nuclear protein that interacts with the fragile X mental retardation (FMR1) protein. Hum. Mol. Genet. 8(13):2557– 66 12. Barker GC. 2002. Microsatellite DNA: a tool for population genetic analysis. Trans. R. Soc. Trop. Med. Hyg. 96(Suppl. 1):21–24 13. B ear M F , H uber K M , Warren S T . 2004. T he m G lu theory of F ragile X mental retardation. Trends Neurosci. 27(7):370–77 14. Berger R, Bloomfield CD, Sutherland GR. 1985. Report of the committee on chromosome rearrangements in neoplasia and on fragile sites. Cytogenet. Cell. Genet. 40(1–4):490– 535 15. Brouwer JR, Mientjes EJ, Bakker CE, Niewenhuizen IM, Severijnen LA, et al. 2006. Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylated Fragile X full mutation. Exp. Cell Res. 313(2):244–53 16. B rown V , Jin P, C eman S, D arnell J C , O ’ D onell W T , et al. 2001. M icroarray identification of F M R P -associated brain m R N As and altered m R N A translational profiles in F ragile X syndrome. Cell 107(4):477–87 17. Ceman S, Brown V, Warren ST. 1999. Isolation of an FMRP-associated messenger ribonucleoprotein particle and identification of nucleolin and the fragile X-related proteins as components of the complex. Cell. Biol. 19:7925–32 18. Ceman S, Nelson R, Warren ST. 2000. Identification of mouse YB1/p50 as a component of the FMRP-associated mRNP particle. Biochem. Biophys. Res. Commun. 279(3):904–8 19. Ceman S, O’Donnell WT, Reed M, Patton S, Pohl J, Warren ST. 2003. Phosphorylation influences the translation state of FMRP associated polyribosomes. Hum. Mol. Genet. 12:3295–305 20. Chen L, Yun SW, Seto J, Liu W, Toth M. 2003. The Fragile X mental retardation protein binds and regulates a novel class of mRNAs containing U rich target sequences. Neuroscience 120(4):1005–17 21. Chiurazzi P, Pomponi MG, Pietrobono R, Bakker CE, Neri G, et al. 1999. Synergistic effect of histone hyperacetylation and DNA demethylation in the reactivation of the FMR1 gene. Hum. Mol. Genet. 8(12):2317–23 www.annualreviews.org • Fragile X Syndrome

7. K ey report showing the R N A -binding properties of F M R P and giving the fi rst insight into its molecular function.

13. A n overview of the role of F M R P in synaptic plasticity where the authors present a theory that can account for diverse neurological and psychiatric aspects of F X S. 16. T he fi rst identification of candidate m R N As regulated by F M R P in mouse brain.

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28. T he latest review on the growing nu mber of diseases k nown to be caused by trinucleotide repeat expansions since the discovery of the causative m utation of F X S.

39. Places F M R P in the postsynaptic compart ment implicating it in local protein synthesis.

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66. F irst report showing the in vivo interaction between mam malian F M R P and both micro R N As and the components of the micro R N A pathway.

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A nnual R eview of G enomics and H u man G enetics


C ontents

Volume 8, 2007

H uman E volution and Its Relevance for G enetic E pidemiology Luigi Luca Cavalli-Sforza ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 1

G ene D uplication: A D rive for Phenotypic D iversity and C ause of H uman D isease Bernard Conrad and Stylianos E. Antonarakis ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 17 D N A Strand Break Repair and H uman G enetic D isease Peter J. McKinnon and Keith W. Caldecott ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 37 T he G enetic L exicon of D yslexia Silvia Paracchini, Thomas Scerri, and Anthony P. Monaco ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 57

Applications of R N A Interference in M ammalian Systems Scott E. M artin and N atasha J. Caplen ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 81 T he Pathophysiology of F ragile X Syndrome Olga Penagarikano, Jennifer G. Mulle, and Stephen T. Warren ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 109

M apping, F ine M apping, and M olecular D issection of Q uantitative Trait L oci in D omestic Animals M ichel Georges ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 131 H ost G enetics of M ycobacterial D iseases in M ice and M en: F orward G enetic Studies of B C G -osis and Tuberculosis A. Fortin, L. Abel, J.L. Casanova, and P. G ros ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 163

C omputation and Analysis of G enomic M ulti-Sequence Alignments M athieu Blanchette ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 193

microR N As in Vertebrate Physiology and H uman D isease Tsung-Cheng Chang and Joshua T. Mendell ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 215

Repetitive Sequences in C omplex G enomes: Structure and E volution Jerzy Jurka, Vladimir V. Kapitonov, Oleksiy Kohany, and M ichael V. Jurka ! ! ! ! ! ! ! ! 241 C ongenital D isorders of G lycosylation: A Rapidly Expanding D isease F amily Jaak Jaeken and Gert M atthijs ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 261 v

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Annotating N oncoding R N A G enes Sam G riffiths-Jones ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 279 U sing G enomics to Study H ow C hromatin Influences G ene Expression Douglas R. H iggs, Douglas Vernimmen, Jim Hughes, and Richard Gibbons ! ! ! ! ! ! ! ! ! 299

M ultistage Sampling for G enetic Studies Robert C. Elston, D anyu Lin, and Gang Zheng ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 327 Annu. Rev. Genom. Human Genet. 2007.8:109-129. Downloaded from arjournals.annualreviews.org by EMORY UNIVERSITY on 08/31/09. For personal use only.

T he U neasy E thical and L egal U nderpinnings of L arge-Scale G enomic Biobanks H enry T. G reely ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 343

I ndexes C umulative Index of C ontributing Authors, Volumes 1–8 ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 365 C umulative Index of C hapter T itles, Volumes 1–8 ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 368 E rrata An online log of corrections to Annual Review of Genomics and Human Genetics chapters may be found at http://genom.annualreviews.org/

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