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History of genetic disease: The molecular genetics of Huntington disease - a history
Author: Gillian P. Bates
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"� 2005 Nature Publishing Group HISTORY OF GENETIC DISEASE The molecular genetics of Huntington disease ? a history Gillian P. Bates Abstract | The Huntington disease gene was mapped to human chromosome 4p in 1983 and 10 years later the pathogenic mutation was identified as a CAG-repeat expansion. Our current understanding of the molecular pathogenesis of Huntington disease could never have been achieved without the recent progress in the field of molecular genetics. We are now equipped with powerful genetic models that continue to uncover new aspects of the pathogenesis of Huntington disease and will be instrumental for the development of therapeutic approaches for this disease. Huntington disease is a late-onset neuro- degenerative disorder that follows an auto- somal-dominant pattern of inheritance. It affects individuals from childhood to old age and follows a course that lasts 15?20 years 1 . There are no sporadic forms of the disease and, in most cases, symptoms present in mid-life and include psychiatric distur- bances, motor impairments and a cognitive decline. Since the late 1970s, the scientific community and lay organizations have worked together to ensure that Huntington disease is at the forefront of new develop- ments. In 1983 the Huntington disease gene (HD) was the first to be mapped to a human chromosome without any prior indication of the gene location. Over the following 10 years, some of the most innovative labo- ratories joined efforts to apply emerging gene-mapping and genomics technologies, which led to the identification of the HD mutation as a CAG-repeat expansion in 1993. The molecular approaches that have been applied to unravel the genetic basis of Huntington disease have been consistently successful. We have gained insights into the cellular mechanisms in which the HD protein participates and the mechanisms through which pathogenic mutations in the HD gene exert their deleterious effects. It is now a good time to review the his- tory of these achievements as we face the next challenge, which involves the transla- tion of basic research into effective treat- ments uniF6AETIMELINEuniF6AF. I begin with an overview of the early history of the disease and the description of its clinical symptoms. I then discuss the approaches and people that suc- cessfully led to the mapping and cloning of the HD gene during the 1980s and early 1990s. The identification and analysis of the HD mutation explained the genetic basis of Huntington disease and also allowed rapid and accurate predictive testing for this dis- ease: sections of this article are devoted to both of these aspects and their ethical impli- cations. Our understanding of the function of the HD protein and some of the insights that we have gained into the molecular pathogenesis of Huntington disease through the generation and analysis of animal models of the disease are then presented. I conclude by discussing the approaches that are being used to develop therapies. In the future, the ability to predict the individuals who will develop Huntington disease must be turned to an advantage through the development of treatments that can substantially delay the onset of symptoms. Genetics and symptoms Although George Huntington was not the first to describe Huntington disease, his 1872 paper was so comprehensive that it was widely acclaimed from the outset and soon gained him international recognition 2 . His father and grandfather were physicians, practising in a rural community in New England, and as a child Huntington often accompanied them on their professional rounds. The collective experience of these 3 generations spanned 78 years and gave him a unique perspective on this hereditary disease. He delivered his paper ?On chorea? before the Meigs and Mason Academy of Medicine at Middleport, Ohio, on 15 February 1872 at the age of 22 uniF6AEREF. 3uniF6AF (FIG. 1). In this paper he described all the clinical features of the adult-onset form of the disease that we recognize today (which is also known as Huntington?s chorea) and he clearly outlined its autosomal-dominant pattern of inherit- ance. The mechanism underlying his obser- vation was not appreciated until after the rediscovery of Mendel?s work 4 in 1900, when it was recognized that Huntington disease probably followed a Mendelian dominant pattern of inheritance 5 . When describing the symptoms, George Huntington noted that the movement disor- der is accompanied by personality changes (nervous excitement) and a cognitive decline (tendency to insanity). He also noticed that families that suffer from Huntington disease were characterized by an increased incidence of suicide, thereby he described many aspects of the adult disease. George Huntington did not encounter the juvenile form of Huntington disease dur- ing his work. This form, which is defined as onset before the age of 21 years, was first described by J. Hoffman using data from a three-generation family. Hoffman identified 2 daughters with onset at 4 and 10 years who showed rigidity, hypokinesia and seizures 6 . This was the first observa- tion showing that the juvenile form of the disease could manifest with clinical features 766 | OCTOBER 2005 | VOLUME 6 www.nature.com/reviews/genetics PERSPECTIVES � 2005 Nature Publishing Group that were strikingly different from those seen in the adult-onset disorder 2 . In 1998 extensive analysis of the ?Huntington disease roster? US database revealed that the HD gene shows ANTICIPATION, but only on paternal inherit- ance 7 , with the consequence that juvenile cases of Huntington disease inherit the disease from their father. Since the clon- ing of the HD gene, mutation analysis has begun to reveal the molecular basis of this gender bias 8,9 , although the mechanism is not understood (see below). Mapping the HD gene In the late 1970s, David Housman suggested to Allan Tobin, who was the scientific direc- tor of the Hereditary Disease Foundation (HDF), that new developments in the field of molecular genetics might facilitate the mapping of human disease genes. Following this conversation, an HDF workshop uniF6AEBOX 1uniF6AF was organized to discuss different strate- gies for mapping the HD gene 10 . Mapping human genes and the generation of linkage maps were not new concepts, but there were few available genetic markers that could be applied to the human genome 11 . This situa- tion changed in 1978 after the identification of RESTRICTION FRAGMENTuniF6BALENGTH POLYMORPHISMS (RFLPs) close to the ?-globin gene 12 , which revealed polymorphic variation in DNA sequence between individuals. Although the extent of this variation was unknown, it was soon proposed that RFLPs might occur suffi- ciently frequently within the genome to allow the generation of DNA-based linkage maps of human chromosomes and the mapping of disease genes 13 . David Housman and James Gusella initiated the first HD gene-mapping project using RFLPs at the beginning of the 1980s. Although the most successful strategy and the length of time required for the completion of this project were subjects for intense debate, it was apparent that large well-characterized families with Huntington disease were required. Fortunately, a large family with Huntington disease had already been found in Venezuela. Nancy Wexler had made some explora- tory visits to Venezuela in the late 1970s because of reports of a high incidence of Huntington disease in the communities of San Luis, Barranquitas and Laguneta, around the shores of Lake Maracaibo. She headed the first research expedition to Maracaibo in 1981 with the aim of identify- ing individuals that were homozygous for the HD gene 14 , and of generating the resources necessary to implement the mapping project. Because it was unclear whether Huntington disease was a HETEROGENEOUS disease, it was advantageous to study a large family emanat- ing from a single founder, thereby ensuring that all affected individuals carried the same mutation in the same gene. The team estab- lished detailed pedigrees and made a neuro- logical and cognitive assessment of affected family members and at-risk relatives. They also took blood samples to send back to the Gusella laboratory for DNA isolation and the establishment of cell lines. Remarkably, and contrary to all expectations, using the Venezuela kindred and a large Ohio fam- ily, the Gusella laboratory found linkage to Huntington disease with the eighth poly- morphic marker tested (G8), which mapped the gene to the short arm of chromosome 4 uniF6AEREF. 15uniF6AF. This was the first genetic disease locus to be mapped to a chromosome with- out any prior knowledge of its chromosomal location. Nancy Wexler recruited a dedicated team for the Venezuela expeditions, who contin- ued the research for the following 20 years 16 , until the recent political situation made Timeline | Benchmarks in Huntington disease research Hoffman describes juvenile Huntington disease (HD) 5 . Mendel?s work is rediscovered 3 . Restriction fragment-length polymorphisms (RFLPs) are first described 12 . The HD gene is mapped to the short arm of chromosome 4 (REF. 15). (1989?1991) Linkage disequilibrium indicates a 2 Mb candidate region 21?23 . The HD gene is isolated and a CAG repeat mutation is identified 26 . Punnet cites HD as autosomal dominant 4 . The Venezeula project is initiated 10 . (1987?1991) Genetic and pulsed maps are refined 17?20,24,113 . Clone contigs of the candidate region are established 116,117 . WFN/IHA, World Federation of Neurology and the International Huntington Association. Exon trapping is developed 25 . The Working Group on HD of the WFN/IHA publishes guidelines on counselling for predictive testing 89 . Max Perutz publishes a paper on polar zippers 55 . The first mouse model for HD is described 58 . Aggregates are described in mouse and patient brains 59,60 . Transcriptional dysregulation is first proposed 61 . An inducible mouse model of HD is described 67 . The first phase-III clinical trials for HD are published 107 . The first high-throughput screen is published 104 . Figure 1 | George Huntington as a young man. Reproduced from the ?Huntington number? of Neurographs 118 . This was the title page of George Huntington?s 1872 paper in the Medical and Surgical Reporter. George Huntington?s paper is published 2 . 1872 1888 1900 1908 1978 1981 1983 1987 1989 1991 1993 1994 1996 1997 1998 2000 2001 2002 NATURE REVIEWS | GENETICS VOLUME 6 | OCTOBER 2005 | 767 FOCUS ON REPEAT INSTABILITY � 2005 Nature Publishing Group it too dangerous to continue. The Venezuela kindreds now encompass 18,149 individuals spanning 10 generations, of whom 15,409 are living 16 . This is a unique longitudinal study of a non-medicated population that has mostly arisen from a single founder and that shows a wide range of clinical variability. These well-characterized kindreds will be an invaluable resource for the identification of genes that modify the onset and progression of Huntington disease. Cloning the HD gene Mapping the HD gene paved the way for isolat- ing the gene, identifying the disease-causing mutation and uncovering the molecular pathogenesis of the disease. However, G8 turned out to be 4 cM (approximately 4 Mb) away from the HD gene 17,18 . The technol- ogy required for gene isolation did not exist in 1983. Few polymorphic DNA markers, which were restricted to BIALLELIC RFLPs, were available. The amount of DNA that could be contained in genomic DNA clones was lim- ited, and the presence of repetitive sequences and/or sequences that were difficult to clone made it impossible to walk along a chromo- some for more than 100?200 kb. Essentially, the entire field of gene cloning and genomics had still to be invented. To facilitate the gene-cloning effort, the HDF invited laboratories developing exper- tise or resources to join forces in a formal col- laboration, and organized regular workshops to facilitate the free exchange of materials and ideas between the group members. This col- laboration was a novel approach in the field of human genetics, as in the mid 1980s the research projects that involved mapping and cloning of human disease genes tended to be highly competitive. The gene was eventually cloned in 1993 by the Huntington disease col- laborative group, which was composed of six laboratories headed by Francis Collins, Jim Gusella, David Housman and John Wasmuth in the United States and Peter Harper and Hans Lehrach in the United Kingdom. Other groups led by Rick Myers, David Cox and Michael Hayden also contributed to the cloning efforts. Although genetic linkage analysis placed the HD gene between G8 and the telomere 17 , this type of analysis could not accurately define the precise position of the gene within this region. To further pinpoint the location of the gene, strategies were used to take advantage of recombination events that had occurred within this chromosomal region in individuals with Huntington disease. The aim of these approaches was to posi- tion the HD gene on one side or the other of the recombination events; however, such events were rare and the strategy crucially depended on an accurate diagnosis. By 1989 four such events had been identified: three of them placed the gene close to the 4p telo- mere, whereas the fourth one predicted a location closer to G8 uniF6AEREF. 19uniF6AF. Cloning the 4p telomere defined the end of the chromo- some and indicated that the highly repeti- tive 100 kb of DNA within the telomeric candidate region was an unlikely place for the gene 20 . To determine which polymor- phisms were in LINKAGE DISEQUILIBRIUM (LD) with HD, studies were conducted to analyse the frequency with which specific alleles of the polymorphisms that were genetically linked to Huntington disease were inherited with the HD mutation. The LD analysis also indicated that the gene was most likely to reside in a 2-Mb region closer to G8 than to the telomere 21?24 . Following the cloning of the gene, it was revealed that the three individuals with putative recombination events pointing to the telomeric location did not have Huntington disease. Various genetic and physical mapping and cloning strategies were combined to define and clone the HD candidate region uniF6AEBOX 2uniF6AF. A novel EXON TRAPPING approach was used to extract exons from cosmids that contained genomic DNA from this 2-Mb region 25 . Trapped exons were used to screen cDNA libraries to isolate genes that were dubbed as ?interesting transcripts? (ITs) by the Gusella and MacDonald group. IT15 and IT16 together comprised most of a 10,366-bp transcript stemming from a large gene that came to be known as IT15. The gene con- tained a CAG-triplet repeat in exon 1 within the ORF that was polymorphic on normal chromosomes and expanded on HD-mutated chromosomes 26 . The causative mutation Triplet-repeat mutations were unknown until 1991, when a CCG expansion in the 5? UTR of the fragile X mental retardation 1 (FMR1) gene was found to cause FRAGILE X SYNDROME 27 (see also Online links box) and a CAG expansion in the ORF of the andro- gen receptor was found to cause SPINAL AND BULBAR MUSCULAR ATROPHY 28 (see also Online links box). In 1992 a CTG expansion in the 3? UTR of the myotonic dystrophy protein kinase (DMPK) gene was found to cause MYOTONIC DYSTROPHY 29 (see also Online links box). Within weeks of the publication of the HD mutation the size of the CAG repeat was being determined in unaffected populations and those affected by Huntington disease in genetic clinics around the world, and genotype?phenotype correlations began to emerge 8,30,31 . Over the following 3 years the Huntington disease-associated alleles and unaffected CAG-repeat distributions were defined. The unaffected range is (CAG) 6?35 repeats. Alleles of (CAG) 40 and above are fully penetrant and cause Huntington Box 1 | Extraordinary people that influence research into Huntington disease Research into Huntington disease has been accelerated owing to the involvement of remarkable people working in the non-professional organizations. In the 1960s two foundations were established in the United States that had a major impact for patients with Huntington disease, by promoting scientific research, care provision and raising the profile of Huntington disease in the community. The Committee to Combat Huntington?s Disease (now the Huntington?s Disease Society of America (HDSA)) was founded in 1967 by Marjorie Guthrie, the widow of the folk singer Woody Guthrie who suffered from Huntington disease and died at the age of 55. Marjorie Guthrie promoted the formation of similar non-professional organizations in other countries, which resulted in the establishment of the International Huntington?s Association (IHA) in 1978. The second organization was the Hereditary Disease Foundation (HDF), which was founded by Milton Wexler in 1968 after his wife was diagnosed with Huntington disease and continued by his daughter Nancy Wexler, who is the current president. Milton Wexler brought a unique approach to helping scientific research with the aim of finding potential treatments or a cure 10 . He organized small workshops with an informal structure in which energetic and creative scientists were invited to participate in free-association discussions to explore new mechanisms that might underlie the aetiology and progression of Huntington disease. The HDF and HDSA were both supported by the exceptional Dennis Shea, who raised US$1 million for the Care and Cure of Huntington?s Disease Foundation at a single dinner in New York in 1989. Since the identification of the Huntington disease gene (HD), Nancy Wexler established the Cure HD Initiative of the HDF, and Barbara Boyle provided the energy and expertise to drive the Coalition for the Cure programme of the HDSA. More recently, the High Q Foundation has established important resources and initiated programmes that are devoted to developing a cure for Huntington disease. 768 | OCTOBER 2005 | VOLUME 6 www.nature.com/reviews/genetics PERSPECTIVES � 2005 Nature Publishing Group 16 15.3 15.2 15.1 14 13 12 11 Isolation of markers using somatic-cell hybrids, jumping libraries and linking libraries Genetic linkage analysis Clone contigs of a 2 Mb candidate region Exon trapping to isolate expressed sequences cDNA clone isolation Mutation detection Chromosome 4p Linkage disequilibrium Telomere cloning, genomic DNA isolation as YAC and cosmid clone contigs Search for polymorphisms Generation of physical maps by pulsed-field gel electrophoresis disease within a normal lifespan, whereas alleles of (CAG) 36?39 confer an increasing risk of developing Huntington disease 32,33 . There is an inverse relationship between the age of onset of Huntington disease and the CAG- repeat size, with alleles of (CAG) 70 repeats and above invariably causing a juvenile onset 8,9,26 . The largest repeat that has been reported so far had approximately (CAG) 250 uniF6AEREF. 34uniF6AF, although alleles of (CAG) 80 and above are extremely rare 8,9 . From the outset it was clear that the CAG-repeat size was not the only determinant of the age of onset, and it was estimated to contribute 50?77% to the variance in different populations 8,30,31 . A recent study of the Venezuela pedigree showed that 59% of the residual variabil- ity in the age of onset can be attributed to genetic or shared environmental factors 16 . LD studies have shown that DNA variation that is close to the GluR6 kainate receptor contributes significantly to this residual variation among individuals with mid- life onset 35,36 . The GluR6 kainate neuro- transmitter receptor binds the excitatory amino-acid glutamate, and this finding supports the idea that EXCITOTOXICITY con- tributes to the pathogenesis of Huntington disease. The CAG repeat is unstable and changes size during transmission from one genera- tion to the next 8,26 . Although this instabil- ity and high variability is independent of paternal or maternal inheritance, large expansions are more likely to occur during male transmission. This is reflected in the large variation in repeat size in sperm 8,37 . The fact that large expansions occur during male transmission and that the age of onset of Huntington disease is inversely related to the size of the CAG-repeat expansion explains why juvenile patients almost exclusively inherit the disease gene from their fathers 8,38 . However, the molecular mechanism that causes large expansions to occur during male gametogenesis remains unknown. The existence of de novo mutations in HD is another issue that had long been debated. Mutation analysis resolved this argument as it clearly showed that non-pathogenic alle- les in the high normal range ((CAG) 27?35 ) could expand into the pathogenic range 39 . It is now known that (CAG) 27?35 alleles can be unstable during transmission 33,40 and extensive analysis has predicted a relatively high mutation rate for HD of ?10% in each generation 41 . Instability of the CAG repeat in somatic tissues has been also reported. Small changes in repeat size were first detected in 1994 uniF6AEREF. 42uniF6AF, but large expansions have recently been demonstrated in brain tissue 43 . The cause of these somatic expansions and their potential role in influencing the onset of disease remains to be determined. Huntingtin and its self association The HD gene encodes a large protein (hunt- ingtin) of 348 kDa in which the CAG repeat is translated into a stretch of polyglutamine residues. It is expressed ubiquitously throughout the CNS, peripheral tissues and during embryonic development 44?48 . In 1993 amino-acid sequence searches revealed no homology to sequences held in protein databases and therefore gave no further insights into the function of huntingtin 26 . In 1995 three independent groups engineered knockout mice and showed that huntingtin is essential for embryonic development as null mice die during embryogenesis 49?51 . Around the same period, it was shown that huntingtin contains HEAT repeats ? Box 2 | Cloning the HD gene After the Huntington disease gene (HD) was mapped in 1983, it was essential to develop approaches that would increase the chance of isolating DNA clones from the short arm of chromosome 4p. The first approach was to develop SOMATICuniF6BACELL HYBRID (and later RADIATION HYBRID) panels that contained the human chromosome region of interest in a rodent cell background. This approach can be used to create a physical map of the donor genome and helped to localize DNA segments that were near the HD gene 108?110 . Jumping library technology was also developed to isolate DNA fragments that lay a few hundred kilobases away from a starting marker 111?113 . This technology was based on the isolation of fragments at the ends of RARE CUTTER RESTRICTION FRAGMENTS (used in PULSEDuniF6BAFIELD GEL ELECTROPHORESIS) and was complemented by LINKING LIBRARIES 114 . It was therefore possible to move along a chromosome by jumping from one end of a rare cutter restriction fragment to the other, crossing the restriction site in the linking library and then jumping to the end of the adjacent fragment 111,113 . Restriction fragment-length polymorphisms (RFLPs.sc), VARIABLE NUMBER OF TANDEM REPEATS LOCI (VNTRs.sc) and dinucleotide repeat polymorphisms were used to generate linkage maps 17,18 . Pulsed-field gel electrophoresis was used in parallel to generate physical maps 24,113 . Linkage disequilibrium and mapping relative to rare recombination events were used to position the HD gene more precisely within this physically defined candidate region 19,21?23 . The ability to clone large fragments of DNA (hundreds of kilobases) as YACs 115 transformed the entire field of gene cloning. The DNA within the HD candidate region was isolated as a YAC CLONE CONTIG 116 , which was used to generate a 2 Mb cosmid DNA contig 117 . Exon trapping was developed to efficiently isolate expressed sequences from total genomic DNA 25 . The isolated cosmids were put through the exon-trapping system and the exons that were identified were used to screen cDNA libraries, which led to the isolation of the HD (IT15) gene 26 . NATURE REVIEWS | GENETICS VOLUME 6 | OCTOBER 2005 | 769 FOCUS ON REPEAT INSTABILITY � 2005 Nature Publishing Group sequences of around 40 amino acids that form hydrophobic ?-helices, which assemble into an elongated superhelix 52 . This structure indi- cated that huntingtin is a large multifunctional scaffold protein 53 . To identify the proteins that interact with huntingtin, the yeast two-hybrid (Y2H) system has been widely used 53 . More recently, a huntingtin protein?protein inter- action network map comprising 186 proteins has been compiled using a high-throughput Y2H approach 54 . In 1994 Max Perutz predicted that poly- glutamine stretches could self-associate to form cross ?-sheet structures, which he called polar zippers, by hydrogen bond- ing between side-chain and main-chain amides 55 (FIG. 2). This was an intriguing proposal because this is the structure of the amyloid deposits that are found in many late-onset neurodegenerative disorders including Alzheimer disease, Parkinson disease and the prion-associated diseases. Three years later it was shown that the protein encoded by exon 1 of the HD gene, which contains polyglutamine repeats in the pathogenic range, could spontaneously aggregate into amyloid fibrils in vitro 56 . Since then, there has been much debate about the stages within the misfolding and aggregation pathway that are pathogenic 57 . Genetic models of Huntington disease In 1996 the first mouse model of Huntington disease was generated in which exon 1 of the HD gene was expressed under the control of human HD promoter sequences 58 . One year later, neuropathological analysis of the mouse brains revealed the presence of ubiquitylated proteinaceous aggregates 59 , which were sub- sequently detected in the brains of patients with Huntington disease 60 . In addition, the analysis of neurotransmitter receptor levels in these mice provided the first indication that transcriptional dysregulation might be an early event in the pathogenesis of Huntington disease 61 . Since then, several transgenic mouse models that express fragments of the human HD gene or the full-length human HD gene have been generated 62,63 . ?Knockin? models, in which a pathogenic CAG repeat has been introduced into the mouse homologue of the HD gene (Hdh) 62,63 , and a transgenic rat model 64 , have also been analysed. Mouse models have been valuable for predicting the neuropathological and molecular changes that occur in the brains of patients with Huntington disease, and they have recently led to useful insights into the peripheral pathogenesis of Huntington disease through identification of muscle and possibly pancreatic pathologies 65,66 . In 2000 Ai Yamamoto created an inducible mouse model in which exon 1 of the HD gene with (CAG) 94 repeats was expressed under the control of a promoter that could be switched off after adding doxycycline to the drinking water 67 . This remarkable study revealed that if symptoms were allowed to develop and then the gene was switched off, huntingtin aggregates were rapidly cleared from the mouse brains and the motor symptoms were reversed. Most importantly, it provided the first indication that treatment in the early stages of Huntington disease might be able to reverse the clinical symptoms. Perhaps surprisingly, it has been pos- sible to model Huntington disease in many single-cell and invertebrate systems includ- ing Saccharomyces cerevisiae 68 , mammalian cells 69,70 , Caenorhabditis elegans 71?73 and Drosophila melanogaster 74?76 . It is remark- able that the CAG-repeat threshold that separates the unaffected and pathogenic repeat ranges is maintained in C. elegans 77 . One consistent finding from these models is that overexpression of chaperone proteins, which help to maintain polyglutamine in a soluble form, is beneficial 68,72,76,78,79 . Enhancer and suppressor screens have proved useful for identifying genes that modify poly- glutamine toxicity in S. cerevisiae 80,81 and D. melanogaster 76 , and a small interfering RNA (siRNA) screen has been used to identify genes that modify polyglutamine aggregation in C. elegans 82 . The genetic reduction of SIN3A, a co-repressor protein that is a component of histone deacetylases (HDAC) complexes, and the use of HDAC inhibitors for genetic and pharmacological approaches show that inhibition of tran- scriptional repressor complexes can rescue D. melanogaster phenotypes 83 . Predictive testing Mapping the HD gene to chromosome 4 in 1983 paved the way for pre-symptomatic and prenatal testing using linkage analysis; however, these tests required DNA sam- ples to be obtained from several family members 84?87 . After the cloning of the gene, accurate testing required only DNA from the at-risk individual. Predictive testing presented choices that had not previously been available to patients, but it also raised significant ethical issues. Perhaps initially surprising, both positive 86 and negative 88 outcomes could cause tremendous upheav- als within families because of feelings of guilt, and through challenging perceived roles and family dynamics. International guidelines to recommend procedures for predictive testing were pre- pared by the lay members and scientific groups of the International Huntington Association (IHA) and Working Group on Huntington disease of the World Federation of Neurology (WFN) after extensive consultation. These guidelines were first published in 1989 and revised in 1994 uniF6AEREF. 89). The recommendations sug- gest that pre-symptomatic testing should only be offered to at-risk individuals who have had the appropriate counselling, are fully informed and wish to proceed. There is now an extensive literature on the impact of pre-symptomatic testing for Huntington disease 90?93 , which has provided the model for establishing pre-symptomatic testing programmes for other diseases. There are also specific situations that pose further ethical dilemmas. First, it has been generally recognized that testing in child- hood for adult-onset untreatable disorders holds the potential of more harm than bene- fit 94 , and the International Guidelines recom- mend against this. Second, there are instances when testing an individual at a 25% risk (for example, a young adult) might inadvertently diagnose a symptom-free at-risk individual (for example, their parent) who does not wish to know their genetic status 95 . Finally, the test also allows prenatal diagnosis for Huntington disease. The uptake of prenatal testing has been relatively low 93,96 , which is most likely due to a combination of factors. The recent advent of pre-implantation genetic testing provides a new option to at-risk couples who wish to avoid the selective termination of pregnancies, even if there is a low success rate and possible complications 97 . The advent of predictive testing also has social, political and economic consequences. Genetic discrimination has become a per- tinent issue over the past decade with the Figure 2 | Max Perutz at the Royal Institution in London in 1994. Photograph taken by A.R. Fersht of the MRC Laboratory of Molecular Biology, Cambridge. 770 | OCTOBER 2005 | VOLUME 6 www.nature.com/reviews/genetics PERSPECTIVES � 2005 Nature Publishing Group advent of genetic testing for many diseases, and is set to become more controversial as the Human Genome Project realizes its predictive potential. Over the past few years, many countries and certain states in the United States have passed legislation to prevent access by employers or insurers to the results of predictive genetic tests 92 . In the United Kingdom the Association of British Insurers (ABI) published an agreement with the government in 2001 that set out a 5-year moratorium on the use of DNA genetic tests by insurers, except when very large sums are insured (over �500,000 for life insurance). This anonymity is important as the adverse financial consequences, and also the fear of genetic discrimination, can lead at-risk indi- viduals to seek anonymous genetic testing (in the United States at least), which is often carried out without the appropriate genetic counselling. Future perspectives Over the past 25 years, our understanding of the molecular pathogenesis of Huntington disease has increased exponentially through the application of molecular genetic approaches and the cloning of the HD gene. However, as was true at the time of George Huntington, there are still no interventions that can be used to halt or slow the progres- sion of Huntington disease, and treatment is limited to managing some of the symptoms. The inducible mouse model has revealed that it might be possible not only to slow down dis- ease progression but in some situations even reverse it. Developing treatments that could slow down or reverse the symptoms would represent a major breakthrough. However, given that predictive testing can identify most individuals who will develop Huntington disease, interventions that might postpone the age of disease onset to beyond a natural lifespan present the most attractive scenario. One therapeutic approach that is currently under development is the use of siRNAs to decrease the level of the huntingtin transcript 98 . Because increasing amounts of mutant HD ? for example, by breeding mouse models to homozygosity ? acceler- ates the onset of a phenotype, it is logical to assume that a decrease in the level of mutant HD should delay the onset of disease symp- toms. Application of this approach relies on the development of a procedure to deliver siRNAs throughout the brain. As the conse- quences of reducing the huntingtin protein levels to less than 50% in the adult brain are unknown, it might be necessary to specifi- cally target the mutant transcript or devise a system that simultaneously delivers normal huntingtin. The problems that are associated with brain delivery make small-molecule thera- peutics an attractive option. Over the past few years, several protocols have been established for screening compounds in genetic models of Huntington disease 99 . Compounds are selected by an hypothesis-driven approach; for example, histone deacetylase inhibitors have been shown to be effective in several model systems including D. melanogaster 83 and the mouse 100?102 . Alternatively, several compounds have been isolated by screen- ing pharmaceutical libraries and have been evaluated in model systems 99,103 . These have been limited to identifying small molecules that inhibit the aggregation of mutant HD by either direct or indirect interac- tions 104?106 . Because phase III clinical trials for Huntington disease are long and expen- sive 107 , it is essential that preclinical trials in mouse models are carried out rigorously and are shown to be reproducible 99 . In par- allel to preclinical testing, the Huntington Study Group (HSG) and more recently the Huntington Project (in the United States) and the Euro-HD Network (in Europe) are investing considerable effort and resources to ensure that the infrastructure necess ary to test promising compounds in the clinic is in place. With such efforts, there is every reason to expect that effective treatments for Huntington disease will become a reality for the next generation. Gillian P. Bates is at the Department of Medical and Molecular Genetics, GKT School of Medicine, King?s College London, 8th Floor Guy?s Tower, Guy?s Hospital, London SE1 9RT, United Kingdom. e-mail: gillian.bates@genetics.kcl.ac.uk doi:10.1038/nrg1686 Glossary ANTICIPATION A phenomenon whereby a disease develops an earlier onset, or more severe symptoms, as it is transmitted through the generations. BIALLELIC A locus at which there are two possible variations of a given DNA sequence that are detectable in the human population. CLONE CONTIG A linear series of DNA clones with overlapping inserts. EXCITOTOXICITY The over-stimulation of excitatory neurotransmitter receptors, which causes an influx of calcium in the postsynaptic neuron. EXON TRAPPING A specialized vector containing splice sites that will splice to and isolate exons that are contained within the genomic insert. FRAGILE X SYNDROME The most common form of human X-chromosome-linked mental retardation that is associated with a folate-sensitive fragile site at Xq27.3. HETEROGENEOUS A description of a genetic disease that is caused by mutations in different genes. LINKAGE DISEQUILIBRIUM Non-random association of alleles at genetically linked loci. LINKING LIBRARIES Genomic libraries of rare cutter restriction sites and their flanking DNA. MYOTONIC DYSTROPHY An autosomal-dominant disease with variable symptoms. The mild form exhibits cataracts that develop in mid to old age, the adult form shows myotonia and muscle weakness, and the most severe form is congenital with a high rate of neonatal mortality. Myotonic dystrophy shows pronounced anticipation on maternal inheritance. PULSEDuniF6BAFIELD GEL ELECTROPHORESIS An electrophoretic technique that is used to separate large fragments of DNA (>20 kb and up to 10 Mb) on an agarose gel by periodically changing the orientation of the electrical field that is applied to the gel. RADIATION HYBRID A type of somatic-cell hybrid in which fragments of chromosomes of one cell type are generated by exposure to X-rays and are subsequently allowed to integrate into the chromosomes of a second cell type. RARE CUTTER RESTRICTION FRAGMENT Fragments generated by restriction endonucleases that cut infrequently in the genome either because the recognition sequence is large or because it contains one or more copies of the CpG dinucleotide. RESTRICTION FRAGMENTuniF6BALENGTH POLYMORPHISM A fragment-length variant that is generated through the presence or absence of a restriction- enzyme recognition site. Restriction sites can be gained or lost by base substitutions, insertions or deletions. SOMATICuniF6BACELL HYBRID An artificially constructed cell in which chromosomes have been stably introduced from cells of a different species. SPINAL AND BULBAR MUSCULAR ATROPHY An X-chromosome-linked mild form of motor neuron disease. VARIABLE NUMBER OF TANDEM REPEATS LOCUS A locus that contains a variable number of short tandemly repeated DNA sequences that vary in length and are highly polymorphic. NATURE REVIEWS | GENETICS VOLUME 6 | OCTOBER 2005 | 771 FOCUS ON REPEAT INSTABILITY � 2005 Nature Publishing Group 1. Bates, G. P., Harper, P. S. & Jones, A. L. (eds) Huntington?s Disease (Oxford Univ. Press, Oxford, 2002). 2. Harper, P. S. Huntington?s disease (W.B. Saunders, London, 1996). 3. Huntington, G. On chorea. Med. Surg. 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T., Carle, G. F. & Olson, M. V. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236, 806?812 (1987). 116. Bates, G. P. et al. Characterization of a yeast artificial chromosome contig spanning the Huntington?s disease gene candidate region. Nature Genet. 1, 180?187 (1992). 117. Baxendale, S. et al. A cosmid contig and high resolution restriction map of the 2 megabase region containing the Huntington?s disease gene. Nature Genet. 4, 181?186 (1993). 118. Browning, W. Huntington number. Neurographs 1, 1?164 (1908). Acknowledgements The author wishes to acknowledge the scientists and clinicians who have contributed to the study of Huntington disease over the past 20 years, but whose work it was not possible to include in this brief historical review. Work in the author?s laboratory is sup- ported by the Wellcome Trust, the Hereditary Disease Foundation, the Huntington?s Disease Society of America?s Coalition for the Cure programme and the High Q Foundation. Competing interests statement The author declares no competing financial interests. Online links DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene DMPK | FMR1 | HD MedlinePlus: http://www.nlm.nih.gov/medlineplus/ medlineplus.html doxycycline OMIM: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=OMIM Alzheimer disease | fragile X syndrome | Huntington disease | myotonic dystrophy | Parkinson disease | spinal and bulbar muscular atrophy FURTHER INFORMATION Department of Medical and Molecular Genetics, King?s College London: http://www.kcl.ac.uk/depsta/memoge Euro-HD Network: http://www.euro-hd.net HDbase: http://hdbase.org Hereditary Disease Foundation: http://www.hdfoundation.org High Q Foundation: http://www.highqfoundation.org Human Genome Project: http://www.ornl.gov/sci/ techresources/Human_Genome/home.shtml Huntington Project: http://www.huntingtonproject.org Huntington?s Disease Society of America: http://www.hdsa.org Huntington Study Group: http://huntington-study-group.org Access to this interactive links box is free online. 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