BIMM110 LECTURES 22-23
FRAGILE SITES. X-LINKED MENTAL RETARDATION. TRINUCLEOTIDE REPEAT EXPANSION
National Foundation ACMG
Fragile
X
A. Introduction
1. Fragile sites
chromosome morphology; induction by folate or aphidicolin
mendelian inheritance
incomplete replication or incomplete chromatin condensation ?2. genetic consequences
chromosome breakeage in cancer?
generally no phenotype, except in the Martin-Bell or fragile X syndrome: site at Xq27.3
clinical features: mental retardation, behvioral problems, macroorchidism
B. Cytogenetics
- expression of fraX in affected males, in affected females, in transmitting males and carrier females showing no symptoms
- expression of fraX in human/rodent hybrid cells
C. Genetics
- the fragile X was until a few years ago unique among the genetic phenomena in man (however, see below)
- the Sherman paradox:
D. Laird's Hypothesis
- a mutated X chromosome must be passed through oogenesis in the female and undergo a cycle of inactivation/reactivation before the full expression of the syndrome is observed;
- not precisely correct, but very stimulating for further studies
E. Molecular Genetics
- linkage to hemophilia A, hemophilia B, G6PD, other cloned X-linked markers
- cloning of the FMR-1 gene by mapping and cloning across the fragile site (positional cloning)
~ 38 kb, 17 exons
- the nature of the mutation: triplet repeat amplification (CGG repeat) in 5'UTR of transcript
- in normal individuals the number of repeats is variable, ranging from 6-50; a pre-mutation can be identified in nonpenetrant transmitting males and unaffected carrier females, where the number of repreats is >60 and <200; affected individuals have a number of repeats often >>200
- it is found that the CGG repeat has some interruptions by an AGG repeat; such AGG repeats appear to stabilize the allele; expansion of the CGG repeat may occur after the loss of an AGG repeat, i.e. the CGG repeat must be >24 perfect repeats before amplification by slippage during DNA replication
- in affected males hypermethylation in upstream CpG island of the gene is observed (promoter region)
- silencing of the gene (no transcript in affected males)- the gene product and its possible function: the FMR-1 protein appears to be an RNA binding protein; however, its target(s) are still unkown
- the FMR-1 protein is ubiquitously expressed, but it is most abundant in the testes and in the brain.
F. Further postzygotic amplification of triplet repeats in somatic cells (??)
in affected males there appears to
be a mechanism (selection, shrinkage) that causes them to have gametes with
the premutation
Triplet repeats and unstable regions of DNA are a more widespread phenomenon with genetic consequences
A. Other Examples
- Martin-Bell or fragile X syndrome
- Huntington's disease (HD), Huntington's
- Spinocerabellar Ataxia (Type I),
- Spinal and Bulbar Muscular Atrophy (AR)
- Myotonic Dystrophy
- Machado-Joseph Disease
FRAXA fragile X syndrome (CGG) n=6-52 in 5'UTR FMR-1 protein lost FRAXE fragile XE mental retardation (GCC) n=7-35 ?? loss of function FRAXF no disorder (GCC) n=29 ?? fragile site FRA16A no disorder (CCG) n=16-49 ?? fragile site FRA11B predisp. to Jacobson syndr. (CGG) n=11 5'UTR fragile site AR (CAG) n=11-33 ORF poly-glutamine track HD Huntington's (CAG) n=9-35 ORF gain of function; dominant; huntingtin with polyglutamine tract DMPK Myotonic Dystrophy (CTG) n=5-35 3'UTR abnormal processing of mRNA for cAMP-dep. muscle protein kinase ??? FRDA Friedreich Ataxia (GAA) n=7-34 intron X25 or frataxin gene; abnormal iron homeostasis; involvement of mitochondria
14 neurological disorders are now known
B. Genetic Aspects:
- still observed: anticipation, i.e. an increase in severity (or a reduction in the age of onset)
- less complicated (?) since most of these loci are autosomal and hence no imprinting (X inactivation) is involved
C. Molecular Genetics:
- most of the genes have been cloned and the encoded proteins have been identified
- mutations (trinucleotide repeats) may or may not be in the coding sequence (ORF);
- polymorphism with regard to the number of repeats is generally observed in a population; there is a normal range (see Table)
- the trinucleotide expansion can affect gene expression, transcript processing, protein function/stability
It continues to be a challenge to understand why an expanded polyglutamine track in the protein huntingtin represents a gain of function, i.e. why is this mutant allele is dominant and almost 100% penetrant.
D. Genetic Screening
A molecular definition of these mutations makes it possible to identify carriers unambiguously by amplifying the trinucleotide repeats by PCR, using the flanking unique sequences as targets for the oligonucleotide primers.
Experience has shown that a large fraction of individuals at risk prefer not to know and refuse the analysis. Since the symptoms of HD appear generally after the child bearing age, screening would have to take place before the symptoms appear in order to inform parents of the potential 50% risk of having an affected child.
Selected references
A. Historical
- Bell, M.V. et.al. (1991). Physical mapping across the
fragile X: Hypermethylation and clinical expression of the fragile X syndrome.
Cell 64, 861-866.
- Craig, I. (1991). Human Genetics: Methylation and the fragile
X. Nature 349, 439-440.
- Fu, Y.-H. et.al. (1991). Variation of the CGG repeat at the
fragile X site results in genetic instability: Resolution of the Sherman paradox.
Cell 67, 1047-1058.
- Hecht, F. (1991). Fragile X gene. Science 253, 1467.
- Heitz, D. et.al. (1991). Isolation of sequences that span the
fragile X and identification of fragile X-related CpG island. Science 251, 1236-1239.
- Kremer, E.J., et.al. (1991a). Mapping of DNA instability at
the fragile X to a trinucleotide repeat sequence p(CCG)n. Science 252, 1711-1714
- Kremer, E.J., et.al. (1991b). Isolation of a human DNA sequence
which spans the fragile X. Am. J. Hum. Genet. 49, 656-661.
- Laird, C.D. (1987) Proposed mechanism of inheritance and expression
of the human fragile X syndrome of mental retardation. Genetics 117: 587-599
- Oberle, I. et.al. (1991). Instability of a 550 basepair DNA
segment and abnormal methylation in fragile X syndrome. Science 252, 1097-1102.
- Sutherland, G.R. (1985) The enigma of the fragile X chromosome
- Sved, J.A. and Laird, C.D. (1990). Population genetic consequences
of the fragile-X syndrome, based on the X-inactivation imprinting model. Am.
J. Hum. Genet. 46, 443-451.
- Verkerk, A.J.M.H., et.al. (1991) Identification of gene (FMR-1)
containing a CGG repeat coincident with a breakpoint cluster region exhibiting
length variation in fragile X syndrome. Cell 65, 905-914.
- Wöhrle, D., Fryns, J.-P., and Steinbach, P. (1990). Fragile
X expression and X inactivation. I. The expression of the fragile site at Xq27.3
is not suppressed on inactive X chromosomes separated from the active homologue.
Hum. Genet. 85, 659-665.
- Yu, W.-D., Wenger, S.L., and Steele, M.W. (1990). X chromosome
imprinting in fragile X syndrome. Hum. Genet. 85, 590-594.
- Yu, S., et.al. (1991) Fragile X genotype characterized by an
unstable region of DNA. Science 252, 1179-1181.
-Hansen, R.S., Canfield, T.K., Lamb, M.M., Gartler, S.M., and
Laird, C.D. (1993). Association of fragile X syndrome with delayed replication
of the FMR1 gene. Cell 73, 1403-1409.
-Morell, V. (1993). The puzzle of the triple repeats. Science
260, 1422-1423.
-Mandel, J.-L., Hagerman, R., Froster, U., Brown, W.T., Jenkins,
E.C., Jacobs, P., Turner, G., Lubs, H., and Neri, G. (1992a). Fifth International
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B. Recent
-Chen, X., Mariappan, S.V.S., Catasti, P., Ratliff, R.,
Moyzis, R.K., Laayoun, A., Smith, S.S., Bradbury, E.M., and Gupta, G. (1995).
Hairpins are formed by the single DNA strands of the fragile X triplet repeats:
Structure and biological implications. Proc. Natl. Acad. Sci. USA 92,
199-5203.
- Dutch-Belgian Fragile X Consortium (1994). Fmr1 knockout
mice: A model to study fragile X mental retardation. Cell 78, 23-33.
- Eicher, E.M. (1994). Sex and trinucleotide repeats. Nature Genet.
6, 221-223.
- Eichler, E.E., Holden, J.J.A., Popovich, B.W., Reiss, A.L.,
Snow, K., Thibodeau, S.N., Richards, C.S., Ward, P.A., and Nelson, D.L. (1994).
Length of uninterrupted CGG repeats determines instability in the FMR1
gene. Nature Genet. 8, 88-94.
- Feng, Y., Zhang, F., Lokey, L.K., Chastain, J.L., Lakkis, L.,
Eberhart, D., and Warren, S.T. (1995). Translational suppression by trinucleotide
repeat expansion at FMR1. Science 268, 731-734.
- Fisch, G.S., Snow, K., Thibodeau, S.N., Chalifaux, M., Holden,
J.J.A., Nelson, D.L., Howard-Peebles, P.N., and Maddalena, A. (1995). The fragile
X premutation in carriers and its effect on mutation size in offspring. Am.
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- Fry, M. and Loeb, L.A. (1994). The fragile X syndrome d(CGG)n
nucleotide repeats form a stable tetrahelical structure. Proc. Natl. Acad. Sci.
USA 91, 4950-4954.
- Hagerman, R.J., Hull, C.E., Safanda, J.F., Carpenter, I., Staley,
L.W., O'Connor, R.A., Seydel, C., Mazzocco, M.M.M., Snow, K., Thibodeau, S.N.,
Kuhl, D., Nelson, D.L., Caskey, C.T., and Taylor, A.K. (1994). High functioning
fragile X males: Demonstration of an unmethylated fully expanded FMR-1 mutation
associated with protein expression. Am. J. Med. Genet. 51, 298-308.
- Hergersberg, M., Matsuo, K., Gassmann, M., Schaffner, W., Lüscher,
B., Rülicke, T., and Aguzzi, A. (1995). Tissue-specific expression of a
FMR1/ß-galactosidase fusion gene in transgenic mice. Hum. Mol.
Genet. 4, 359-366.
- Maddox, J. (1994). Triplet repeat genes raise questions. Nature
368, 685
- Siomi, H., Choi, M., Siomi, M.C., Nussbaum, R.L., and Dreyfuss,
G. (1994). Essential role for KH domains in RNA binding: Impaired RNA binding
by a mutation in the KH domain of FMR1 that causes fragile X syndrome. Cell
77, 33-39.
- Siomi, M.C., Siomi, H., Sauer, W.H., Srinivasan, S., Nussbaum,
R.L., and Dreyfuss, G. (1995). FXR1, an autosomal homolog of the fragile X mental
retardation gene. EMBO J. 14, 2401-2408.
- Warren, S.T. and Ashley, C.T., Jr. (1995). Triplet repeat expansion
mutations: The example of fragile X syndrome. Annu. Rev. Neurosci. 18,
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- Ashley,C.T.Jr., and S.T. Warren (1995) Trinucleotide repeat
expansion and human disease. Annu.Rev.Genet. 29: 703-728
- Helderman-van den Enden, A.T., Maaswinkel-Mooij, P.D., Hoogendoorn,
E., Willemsen, R., Maat-Kievit, J.A., Losekoot, M., and Oostra, B.A. (1999).
Monozygotic twin brothers with the fragile X syndrome: different CGG repeats
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- Hong, Y.K., Ontiveros, S.D., Chen, C., and Strauss, W.M. (1999).
A new structure for the murine Xist gene and its relationship to chromosome
choice/counting during X-chromosome inactivation. Proc.Natl.Acad.Sci.U.S.A
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- Johnston, C.M., Nesterova, T.B., Formstone, E.J., Newall, A.E.,
Duthie, S.M., Sheardown, S.A., and Brockdorff, N. (1998). Developmentally regulated
Xist promoter switch mediates initiation of X inactivation. Cell 94, 809-817.
- Kaufmann, W.E. and Reiss, A.L. (1999). Molecular and cellular
genetics of fragile X syndrome. Am.J.Med.Genet. 88, 11-24.
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- Tassone, F., Hagerman, R.J., Gane, L.W., and Taylor, A.K. (1999).
Strong similarities of the FMR1 mutation in multiple tissues: postmortem studies
of a male with a full mutation and a male carrier of a premutation. Am.J.Med.Genet.
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