d. Genet., Vol. 75, Number 2, August 1996, pp. 193-217. ~) Indian Academy of Sciences REVIEW ARTICLE Dynamic mutations in human genes: a review of trinucleotide repeat diseases JOHN W. LONGSHORE and JACK TARLETON Greenwood Genetic Center, 1 Gregor Mendel Circle, Greenwood, SC 29646, USA MS received 8 April 1996 Abstract. Dynamic mutations in human genes result from unstable trinucleotidc repeats embedded within the transcribed region. The changeable nature of these mutations from generation to generation is in contrast to the static inheritance of other single-gene mutational events, e.g. point mutations, deletions, insertions and inversions, typically associated with Mendelian inheritance patterns, lntergenerational instability of dynamic mutations within families has provided an explanation for the genetic anticipation, leading to increasing severity or earlier age of onset in successive generations, associated with certain inherited disorders. While models for genomic instability presume that trinucleotide repeat expansion results fl'om disruption of the DNA replication process, experimental evidence has not yet been obtained in support of this contention. Nevertheless, examples of unstable trinucleotide repeats continue to increase, although not all are associated with a specificphenotype. Five disorders resulting from small-scale expansions of CAG repeats within the protein-coding region are known: spinobulbar muscular atrophy, Huntington's disease, spinocerebellar ataxia type 1, dentatorubral- pallidoluysian atrophy (DRPLA) and Machado-Joseph disease. A sixth disorder, Haw River syndrome, is allelic to DRPLA. Five folate-sensitive chromosomal fi'agile sites characterized to date, viz. FRAXA, FRAXE, FRAXF, FRAIlB and FRA16& all have large-scale CGG repeat expansion. Two disorders, fragile X syndrome and FRAXE mental retardation, result from instability of CGG repeats in the 5' untranslated region of FMRI and FMR2 genes respectively. FRA11B lies close to chromosome 1lq deletion endpoints in many Jacobsen syndrome patients and may be related to the deletion event producing partial aneuploidy for 1lq. Expansion of FRAXF and FRA16A has not been assodated with a phenotype. Myotonic dystrophy results from a large-scale CTG expansion in the 3' untranslated region of the myotonin protein kinase gene while Friedreieh's ataxia has recently been found to have a large-scale GAA repeat in the first intron of X25. This article reviews the characteristics of trinucleotide repeat disorders and summarizes current understanding of the molecular pathophysiology. Keywords. Dynamic mutations; trinucleotide repeats; microsatellite instability. 1. Introduction During the last five years an extraordinary type of mutational event, dynamic mutation resulting from expansion of trinucleotide repeats embedded in certain genes, has been shown to be involved in a growing number of human neurological disorders. Dynamic mutation is yet another addition to the list of non-Mendelian genetic events that began when McClintock's discovery of transposable elements suggested that genomes are not static. The disorders associated with unstable trinucleotide repeats account worldwide for millions of individuals with physical and mental impairments, and have tremendous social and psychological costs to the affected individuals~ their families, their communi- ties, and governments. The biological basis for the connection between trinucleotide repeat genes and neurological disorders is unknown; so is the molecular basis for instability of only 193 194 John W. Longshore and Jack Tarleton trinucleotide repeats (as opposed to mononucleotide, dinucleotide or tetranucleotide repeats) in human disease. The one common feature of all the trinucleotide repeats that cause human disorders is the presence of the repeats within the transcribed region of genes. The molecular characterization of dynamic mutations has provided an explanation for the high variability and unusual patterns of inheritance found in some human disorders. This new understanding naturally leads to the hope that complex familial inheritance patterns, such as those found with bipolar disorder and manic-depressive psychosis, will eventually be unravelled. In addition, new information on the products of genes containing unstable trinudeotide repeats is likely to provide fresh insights about cellular physiology. In this review we describe the properties of trinucleotide repeats and summarize current understanding of the molecular pathophysiology of the known trinucleotide repeat diseases. Most of the information presented is recent, and more complete for some disorders than for others. For example, substantial information has accumulated regarding fragile X syndrorne but very little is known about Machado-Joseph disease. However, the intriguing nature of trinucleotide repeat mutations and the impact of the disorders on human health are sure to lead to many more investigations and eventually to better understanding of the disease genes and their relation to cellular processes. 2. Types of trinucleotide repeat diseases Unstable DNA sequences composed of repeated trinucleotides were first discovered in 1991 in two X-linked human genes, AR (androgen receptor) (LaSpada et at. 1991) and Table 1. Characterized trinueleotide repeats. Year gene Disorder (involved gene) Locus Product characterized Disorders with CAG repeats in the protein-coding region Spinobulbar muscular atrophy (AR) Xq 13 Androgen receptor 1991 Huntington disease (ITIS) 4p16 Huntingtin 1993 Spinocerebellar ataxia (SCAt) 6p22 Ataxin I993 Dentatorubral-pallidoluysian atrophy (B37) 12p12 Atrophin 1994 Haw River syndrome (B37) Machado-Joseph disease (MJD) 14q23.3 Not named 1994 Disorders associated with fragile sites Fragile X mental retardation (FMR1) FRAXA FMRP 1991 Xq27.3 Fragile X mental retardation (FMR2) FRAXE Not characterized 1993 Xq28 1lq - Jacobsen syndrome (CBL2) FRAllB CBL2 protein 1995 (FRAllB) 11 @3.3 CTG repeat in 3' untranslated region Myotonic dystrophy (MTPK) 19q13 Myotonin protein 1992 kinase GAA repeat within an intron Friedreich's ataxia (X25) 9q13 Frataxin 1996 Characterized sites containing (CGG)~ but producing no known phenotype when expanded FRAXF - Xq28 FRA16A- 16p13.1 Trinucleotide repeat diseases 195 FMR1 (fragile X mental retardation) (Kremer et al. 1991; Oberle et at. 1991; Verkerk er al. 1991;Yu et al. 1991). Instability of the trinucleotide repeat in these genes leads to spinobulbar muscular atrophy (SBMA) and the fragile X syndrome respectively, the first disorders in any species known to result from heritable unstable DNA repeat sequences. Since the discovery of these two disorders, other trinucleotide repeat disorders have been discovered (table 1). In retrospect, AR and FMR1 contain the first examples of two subgroups of trinucleotide repeats: (i) small-scale expansions in CAG repeats (encoding polyglutamine segments) in protein-coding regions (figure 1) and (ii) large-scale expansions in CGG repeats in the untranslated portion of gene transcripts that span all folate-sensitive chromosomal fragile sites ctaaracterized to date (figure 2b). (The folate-sensitive fragile sites are chromosomal constrictions occasionally expressed when cells are cultured in a folate-depleted medium.) In the fragile site-associated group, permutations of the CGG repeat may vary with strand or reading frame (figure 3). The recent characterization of the gene involved in Friedreich's ataxia, which contains the first example of an unstable GAA repeat, documents that other triplet types may also cause instability (Campuzano et al. 1996). The largest group of diseases associated with dynamic mutations involve CAG expansions and disruption of normal neurological processes. In addition to AR, genes containing CAG embedded in the protein-coding region include I T-15 (Huntington's (a) Ib) : Ce) " I -] n t ! u l i Figure 1. Polymerase chain reaction detection of CAG repeats. Primers comprising se- quences that flank the CAG repeat region were used to generate PCR products labelled with 32p. Separation was by electrophoresis on polyacrylamide sequencing gels. Pathological alleles are toward the upper end of the gels (upper bracket) and normal alleles are below (lower bracket). (a) Abnormal HD alleles with 39 to 45 repeats (6 normal controls are included). Normal alleles shown here have 15-25 repeats. (b) DRPLA patient with 66 repeats. Normal alleles shown here have 8-16 repeats. (c) Machado-Joseph disease patient with 77 repeats. Normal alleles shown here have 14-30 repeats. (Autoradiographs provided by Dr Nick Potter, University of Tennessee, Knoxville) 196 John W. Longshore and Jack Tarleton (a) (b) 0 Ot - tlbO= -o ,tt:w o mw o 1 12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 1 2 3 4 5 6 7 8 910 Figure 2. Southern blot detection of fragite X syndrome and myotonic dystrophy patients. (a) CTG expansions in myotonic dystrophy patients. In lanes 1-5, DNA was digested with EcoRI. In lanes 6-10, the same patient samples were digested with BamHI. Lanes 2 and 7 contain DNA from a normal control. The two different enzymes are used to detect the range of expanded alleles. For example, the sample in lanes 3 and 8 can be more precisely sized in the BamHI digest. The arrows indicate normal bands in both digests. The EcoRI digest detects a polymorphism of 8"5-kb and 9-5-kb bands as seen in lane 2. (b) Premutations and full mutations in fragile X patients as assayed by the double restriction enzyme digestion method described in Rousseau et al. 199l.