Molecular Basis of Genetic Instability of Triplet Repeats

Abstract

A veritable explosion is taking place in our understanding of the human genetics, biochemistry, and DNA structural issues related to human hereditary neuromuscular and neurodegenerative diseases. Also, the non-Mendelian expansion process that elicits these disease manifestations (anticipation) is under intense investigation. Within the last 3 years, the molecular basis of 10 human genetic disorders (including fragile X syndrome (FRAXA and FRAXE), myotonic dystrophy (DM), ( 1The abbreviations used are: DMmyotonic dystrophyHDHuntington's diseaseSCA1spinocerebellar ataxia type 1DRPLAdentatorubral-pallidoluysian atrophybpbase pair(s)SBMAspinobulbar muscular atrophy.) Kennedy's disease, Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA1), and dentatorubral-pallidoluysian atrophy (DRPLA)) has been partially established (reviewed in (1.Davies K. Warren S. Genome Analysis. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar, 2.Bates G. Lehrach H. BioEssays. 1994; 16: 277-284Crossref PubMed Scopus (112) Google Scholar, 3.Sutherland G.R. Richards R.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3636-3641Crossref PubMed Scopus (301) Google Scholar, 4.Panzer S. Kuhl D.P.A. Caskey C.T. Stem Cells. 1995; 13: 146-157Crossref PubMed Scopus (27) Google Scholar, 5.Krahe R. Ashizawa T. Wetherall J. Groth D. Hypervariable Genetic Markers. CRC Press, Inc., Boca Raton, FL1995: 29-60Google Scholar)). The diseases are characterized at the molecular level by the expansion of a simple triplet repeat (CTG and CGG) from less than 15 copies of the repeat in normal individuals to scores of copies in affected cases; thousands of copies are found in some cases of fragile X and myotonic dystrophy. These increases in size occur upon passage of an expanded repeat in the chromosome to offspring. Moreover, the symptoms of these diseases follow an unusual genetic pattern called anticipation, in which the disease becomes more severe and has an earlier age of onset with each successive generation (reviewed in (1.Davies K. Warren S. Genome Analysis. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar, 2.Bates G. Lehrach H. BioEssays. 1994; 16: 277-284Crossref PubMed Scopus (112) Google Scholar, 3.Sutherland G.R. Richards R.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3636-3641Crossref PubMed Scopus (301) Google Scholar, 4.Panzer S. Kuhl D.P.A. Caskey C.T. Stem Cells. 1995; 13: 146-157Crossref PubMed Scopus (27) Google Scholar, 5.Krahe R. Ashizawa T. Wetherall J. Groth D. Hypervariable Genetic Markers. CRC Press, Inc., Boca Raton, FL1995: 29-60Google Scholar, 6.Wells R.D. Sinden R.R. Davies K. Warren S. Genome Analysis. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 107-138Google Scholar)). The instability of repeats in the genome has also been linked to hereditary nonpolyposis colon cancer, which may involve mutations in mismatch repair functions(4.Panzer S. Kuhl D.P.A. Caskey C.T. Stem Cells. 1995; 13: 146-157Crossref PubMed Scopus (27) Google Scholar, 5.Krahe R. Ashizawa T. Wetherall J. Groth D. Hypervariable Genetic Markers. CRC Press, Inc., Boca Raton, FL1995: 29-60Google Scholar, 7.Thibodeau S.N. Bren G. Schaid D. Science. 1993; 260: 816-819Crossref PubMed Scopus (2812) Google Scholar, 8.Shibata D. Peinado M.A. Ionov Y. Malkhosyan S. Perucho M. Nat. Genet. 1994; 6: 273-281Crossref PubMed Scopus (455) Google Scholar, 9.Baker S.M. Bronner C.E. Zhang L. Plug A.W. Robatzek M. Warren G. Elliott E.A. Yu J. Ashley T. Arnheim N. Flavell R.A. Liskay R.M. Cell. 1995; 82: 309-319Abstract Full Text PDF PubMed Scopus (476) Google Scholar). myotonic dystrophy Huntington's disease spinocerebellar ataxia type 1 dentatorubral-pallidoluysian atrophy base pair(s) spinobulbar muscular atrophy. For example, Huntington's disease shows anticipation and has expanded CAG triplet repeats(1.Davies K. Warren S. Genome Analysis. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar, 2.Bates G. Lehrach H. BioEssays. 1994; 16: 277-284Crossref PubMed Scopus (112) Google Scholar, 3.Sutherland G.R. Richards R.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3636-3641Crossref PubMed Scopus (301) Google Scholar, 4.Panzer S. Kuhl D.P.A. Caskey C.T. Stem Cells. 1995; 13: 146-157Crossref PubMed Scopus (27) Google Scholar, 5.Krahe R. Ashizawa T. Wetherall J. Groth D. Hypervariable Genetic Markers. CRC Press, Inc., Boca Raton, FL1995: 29-60Google Scholar, 10.Huntington's Disease Collaborative Research GroupCell. 1993; 72: 971-983Abstract Full Text PDF PubMed Scopus (7118) Google Scholar). A CAG repeat of between 11 and 34 bp in the normal population encodes a polyglutamine tract in the IT15 gene. Expansion to about 90 bp occurs in HD patients. The age of onset correlates with the length of the triplet repeat with the largest changes in repeat lengths seen upon paternal transmission(11.Duyao M. Ambrose C. Myers R. Novelletto A. Persichetti F. Frontali M. Folstein S. Ross C. Franz M. Abbott M. Gray J. Conneally P. Young A. Penney J. Hollingsworth Z. Shoulson I. Lazzarini A. Falek A. Koroshetz W. Sax D. Bird E. Vonsattel J. Bonilla E. Alvir J. Conde J.Bickham Cha J.-H. Dure L. Gomex F. Ramos M. Sanchez-Ramos J. Snodgrass S. de Young M. Wexler N. Moscowitz C. Penchaszadeh G. MacFarlane H. Anderson M. Jenkins B. Srinidhi J. Barnes G. Gusella J. MacDonald M. Nat. Genet. 1993; 4: 387-392Crossref PubMed Scopus (904) Google Scholar). Sperm display a heterogeneous expanded repeat length. An intermediate allele, IA, containing 30-38 (or 34-38) repeats (perhaps similar to a premutation in fragile X or DM) has been identified. Initial reports suggest that sporadic expansion of the IA allele occurs only through paternal transmission(12.Goldberg Y.P. Kremer B. Andrew S.E. Theilmann J. Graham R.K. Squitieri F. Telenius H. Adam S. Sajoo A. Starr E. Heiberg A. Wolff G. Hayden M.R. Nat. Genet. 1993; 5: 174-179Crossref PubMed Scopus (235) Google Scholar, 13.Myers R.H. MacDonald M.E. Koroshetz W.J. Duyao M.P. Ambrose C.M. Taylor S.A.M. Barnes G. Srinidhi J. Lin C.S. Whaley W.L. Lazzarini A.M. Schwarz M. Wolff G. Bird E.D. Vonsattel J.-P. G. Gusella J.F. Nat. Genet. 1993; 5: 168-173Crossref PubMed Scopus (227) Google Scholar). The function of the gene product is uncertain(14.Trottier Y. Devys D. Imbert G. Sandou F. An I. Lutz Y. Weber C. Agid Y. Hirsch E.C. Mandel J.-L. Nat. Genet. 1995; 10: 104-110Crossref PubMed Scopus (382) Google Scholar). Considering Mendelian genetic principles, anticipation was an enigma. The discovery of expanding triplet repeats (or "mutable mutations") in diseases showing anticipation afforded a physical basis for this unusual genetic phenomenon. Expansion of the triplet repeat is responsible for the genetic defect, influencing the activity of a glutamine-containing protein (SBMA, HD, SCA1, and DRPLA) or influencing the level of expression of a gene with which the repeat is associated (fragile X and DM)(1.Davies K. Warren S. Genome Analysis. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar, 2.Bates G. Lehrach H. BioEssays. 1994; 16: 277-284Crossref PubMed Scopus (112) Google Scholar, 3.Sutherland G.R. Richards R.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3636-3641Crossref PubMed Scopus (301) Google Scholar, 4.Panzer S. Kuhl D.P.A. Caskey C.T. Stem Cells. 1995; 13: 146-157Crossref PubMed Scopus (27) Google Scholar, 5.Krahe R. Ashizawa T. Wetherall J. Groth D. Hypervariable Genetic Markers. CRC Press, Inc., Boca Raton, FL1995: 29-60Google Scholar). All triplet repeat genetic diseases identified to date show anticipation. Several other diseases also show anticipation including spinocerebellar ataxia type 2(15.Pulst S.-M. Nechiporuk A. Starkman S. Nat. Genet. 1993; 5: 8-10Crossref PubMed Scopus (65) Google Scholar), bipolar affective disorder(16.McInnis M.G. McMahon F.J. Chase G.A. Simpson S.G. Ross C.A. DePaulo Jr., J.R. Am. J. Hum. Genet. 1993; 53: 385-390PubMed Google Scholar), and hereditary spastic paraparesis (Strumpell's disease)(17.Bruyn R.P.M. van Deutekom J. Frants R.R. Padberg G.W. Clin. Neurol. Neurosurg. 1993; 95: 125-129Crossref PubMed Scopus (22) Google Scholar). If a correlation exists between anticipation and triplet repeats, many more diseases showing anticipation may be identified since there are more than 40 genes containing associated triplet repeats. An understanding of the molecular mechanisms of triplet repeat instabilities (expansions and deletions) is important for the comprehension of anticipation. Kang et al.(18.Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar) have established a defined genetic system that shows promise for the dissection of this process. The frequency of genetic expansions or deletions in Escherichia coli depends on the direction of replication(18.Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar). Large expansions occur predominantly when the CTGs are in the leading template strand rather than the lagging strand. However, deletions are more prominent when the CTGs are in the opposite orientation (Fig. 1). Most deletions generate products of defined size classes. Strand slippage coupled with non-classical DNA structures (Fig. 2) probably accounts for these observations and relates to expansion-deletion mechanisms in eukaryotic chromosomes. To study expansions, these workers determined if a plasmid that contains (CTG)130 is completely homogenous as a cloned molecule or if deletions and expansions had occurred that gave rise to sequence heterogeneity, even in a tiny percent of the molecules. The insert containing the triplet repeat was excised from the vector and separated by gel electrophoresis. The regions of the gel either above or below the insert band were eluted and "recloned"; recombinant plasmids were obtained that contained successively larger or smaller inserts, respectively. The family

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