Genetics and molecular biology of Huntington's disease

N. Barden.

46

Pepin, M-C. and Barden,

N. (1991)

Mol.

Cd.

Biol. 11,

50

1647-1653

J. Reul and F. Holrboer

- Antidepressants

and the HPA system

VIEWPOINT

PepIn, M-C., Pothier, F. and Barden, N. (1992) Nature 355, 725-728

47 Se&l, J.R. and Fink, G. (1992) NeuroendodnoZogv 55,621-626 48 Brady, L.S. et al. (1991) J. Clin. Invest. 87,831-837 49 Peiffer, A., VeIIIeux, S. and Barden, N. (1991) Psychoneuroendoninology 16,505~515

51 Stec, I. et al. (1994) J. Psychiatr. Res.28, 1-S 52 Richard, D. et al. (1993) Am. J. Physiol. 265, 146-150 53 Pepin, M-C., Pothier, F. and Barden, N. (1992) Pharmacol. 42, 991-995

Mol.

PERSPECTIVES 0~ DISEASE

Genetics and molecular Huntington’s disease

biology

of

RogerL. Albin and Danilo A. Tagle In 1993, the genetic

abnormality

responsible

for Huntington’s

disease was identified

as a tri-

nucleotide-repeat expansion in a novel gene. Much has been learned about the molecular genetics of Huntington’s disease and the possible effects of the trinucleotide expansion in the development is widely

of this disease and other

expressed

throughout

neurological

disorders.

The Huntington’s

the brain and in many non-neural

disease locus

tissues. Current

speculation

about the pathogenesis of neuronal death concentrates on a ‘gain of function’ effect in which the abnormal protein has acquired a new and lethal property. Future research will define the normal

function

of the Huntington’s

of function, and explore abnormal allele. Trends Neurosci. (1995)

the factors

disease locus, test hypotheses that determine

susceptibility

the putative

gain

to the effects of the

18, 11-14

UNTINGTON’S DISEASE (HD) is a completely H penetrant autosomal dominant neurodegenerative disease that is characterized by psychiatric disorders, dementia and involuntary movements’,‘. The typical age of onset of HD is in the fourth and fifth decades of life and the average duration of disease is 15 to 20 yearslt3. Huntington’s disease is incurable, and death results usually from infectious complications of immobility. This disease has an estimated prevalence in North America and Europe of 4-10 out of 100 000 (Refs 1 and 4). The social and emotional impact of HD is disproportionately greater than its prevalence because of its onset in the prime working years, the prolonged and progressive course and the familial nature of the disease. Neuropathologic studies indicate that neuronal loss is especially marked in the striatum with the sparing of some subpopulations of strlatal interneurons, and degeneration of striatal projection neurons in a specific temporal sequencesa. A substantial body of evidence suggests that neuronal death is due to an N-methyl-D-aspartate @MDA)-receptor-mediated excitotoxlc mechanismg~lO. Genetics

The HD locus was one of the first disease-associated loci to be mapped using restriction-fragment polymorphisms l1. This result made presymptomatic detection of HD-allele carriers, and the discovery that HD is a true dominant condition with homozygotes possessing a clinical phenotype identical to 0 1995, Elsevier Science Ltd

neuronal

regarding

that of heterozygotes possible’“13. Despite this early success, the search for the HD gene required approximately ten years of effort. The persistence of the investigators was rewarded in 1993 with the discovery of the locus involved14. Exon amplification of cosmids in the chromosome 4~16.3 interval yielded ‘interesting transcript 15’ (ITlS) from a novel gene in which an expanded CAG-repeat size within the predicted open reading frame was associated with HD chromosomes. The IT15 nucleotide sequence and its predicted protein product (termed huntingtin) lack homology to any previously characterized gene in any portion of the sequence other than the polyCAG tract. Huntington’s disease joined the growing number of inherited diseases, such as fragile-X syndrome, myotonic dystrophy, X-linked spinal-bulbar muscular atrophy (SBMA or Kennedy’s syndrome), spinocerebellar ataxia type I (SCAl) and dentatorubro-pallido-luysian atrophy (DRPLA and related syndromes), that are characterized by expanded trlnucleotide repeats. Genomic sequencing of exon-intron boundaries indicates that IT15 spans 180 kb and contains 67 exons”. The predicted open reading frame yields a projected protein, containing 3144 amino acid residues, with a predicted molecular mass of 348 kDa. Preliminary immunoprecipitation experiments suggest that the molecular mass of huntingtin is approximately 330 kDa, consistent with the molecular weight suggested by the predicted open reading frame16. The sequence of the IT15 transcript and its murine homologue (HDh) T&3 Vol. 18, No. 1, 1995

Roger L. Albin is at the Dept of Neurology, University of Michigan, Ann Arbor, MI 48109, USA, and Danilo A. Tagle is at the National Institutes of Health, National Center for Human Genome Research, Laboratory of Gene Transfer Building 49, Rm 3 A18, 49 Convent Dr., MSC

4470,

Bethesda, MD 20892, USA.

11

PERSPECTIVES 0NDISEASER. Albin

and D. Tagle

- Huntington’s

disease

are remarkably similar with 90% homology in the predicted coding sequence and a high degree of sequence identity in the 5’ and 3’ untranslated regions”,i8. The portion of the murine sequence that corresponds to the polyCAG tract is significantly shorter and is interrupted by a CAA codon’7,‘8. Preliminary studies suggest significant sequence conservation across a variety of mammalian species”,19. Detailed studies, involving large numbers of patients with HD and controls, demonstrated that the IT15 CAG-repeat range of normal chromosomes is 9-39 (mean 18-19 with the vast majority of chromosomes exhibiting repeat lengths shorter than 30), whereas HD chromosomes show repeat lengths of 36-121 (mean 42-46, with the vast majority of chromosomes exhibiting repeat lengths greater than 40)2s24. From these studies, repeat lengths of 40 or more virtually guarantee that carriers (individuals possessing the abnormal allele but without symptoms) will develop HD, while repeat lengths of 30 or less make development of HD very unlikely. In some patients with HD, repeat lengths close to the normal mean have been found. These individuals might represent diagnostic misclassification or sample misidentification”. The future of the very small number of asymptomatic individuals from HD families whose repeat lengths fall in the 31-39 range is uncertain. Repeat lengths vary from generation to generation, with both expansions and contractions occurring and an overall tendency for repeat length to increasez6,“. In fact, mutational bias towards lengthening of alleles might be driving an increase in the prevalence of HD (Ref. 20). The sex of the transmitting parent has a significant influence on magnitude of repeat-length changes. When transmitted from the mother, repeat-length increases or decreases within a range of approximately four CAG units with a mild tendency towards repeat-length increase. When transmitted from the father, there is a much larger range of expansions, up to a doubling of paternal repeat length, and expansions occur much more frequently than contractions26,27. Identical repeat lengths were obtained in DNA from four pairs of monozygotic twins with HD, suggesting that instability of repeat number occurs during gametogenesis or very early embryonic lifez8. Sperm DNA from patients with HD exhibits great variation in repeat length, suggesting that the meiotic events or number of mitoses during spermatogenesis are an important source of repeat-length instability. This is reflected also in the great variation in repeat size seen with paternal transmissionz8. Repeat length has some influence on the age of onset of HD22,24,2q,30. Many individuals with repeat lengths greater than 50 have onset of HD before the age of 30. However, early onset of HD can occur with shorter repeat lengths, and individuals with identical repeat numbers vary significantly in age of onset of HD. While the overall correlation between repeat length and age of onset is quite strong, repeat length accounts for only approximately 50% of the variance in age of onset. Even taking the difficulties of assigning the age of onset of clinically manifest HD into account, these results indicate that other factors, perhaps modifying genes, influence significantly the effects of the abnormal IT15 allele. In addition, repeat length has little correlation with the 12

77h’S Vol. 18, No. 1, 1995

type of symptoms manifested initially by patients with HD (Ref. 29). Several cases of so-called sporadic HD have been described in which the clinical phenotype appears without an established family history. Analysis of these patients has revealed the presence of expanded CAG repeats typical of HD’4,31-33.The father of these affected individuals has been found to harbor an intermediate-range allele with a repeat number between 30 and 40. Presumably, instability of repeat number during transmission has led to an increase in repeat number into the affected range. In view of the large variation in repeat size that occurs during spermatogenesis, it is notable that all documented increases, from intermediate-range alleles to expanded alleles associated with an HD phenotype, have occurred during transmission from the father. The frequency of these events is unknown, and the genetic risk of the progeny of intermediate-allele carriers cannot be estimated presently. IT I5 expression

Initial northern analysis of gene expression indicated that the 10.5 kb IT15 transcript was expressed in many tissues14. Similar patterns of expression are observed for an alternatively polyadenylated 13.7 kb transcript with higher expression of the longer transcript in brain3h36. Subsequent analysis with in situ hybridization in rodent and human tissues has shown that IT15 transcripts are expressed widely in both neural and non-neural tissues with relatively high levels of expression in neurons, testes, ovaries and 1ung34,35.These studies indicate that almost all neurons are labeled with no qualitative difference in IT15transcript expression between neurons of different brain regions. Recent quantitative studies have confirmed this (B. Landwehrmeyer, pers. commun.). In HD striatal sections, expression of IT15 mRNA is decreased as a consequence of neuronal 10ss~~,~‘. The widespread expression of IT15 mRNA in brain and other tissues has been confirmed by a recent immunohistochemical study of the distribution of huntingtin16. The widespread expression of IT15 mRNA militates against one possible explanation of the regional selectivity of HD pathology: selective regional expression of the gene product. Expression of IT15 mRNA is not qualitatively different between striatal neurons and neurons in less affected brain regions, nor is there any evidence of gonadal dysfunction in patients with HD. However, these results do not eliminate the possibility that there could be regionally specific splice variants of huntingtin, and that regional differences in splice-variant expression could play a role in the pattern of pathology. Alternative splicing might occur in the murine homologue of IT15 but is not thought to occur to a major extent with IT15 itself’5,‘7,‘8. An alternative explanation for the regional specificity of pathology in HD would be somatic instability of repeat number. However, no evidence for somatic instability of CAG-repeat number is found in comparisons of DNA from brain and lymphoblasts of the same individuals27,28. A small degree of somatic mosaicism has been reported among brain regions for very long repeat lengths without a good correlation between regional pathology and the

R. Albin

differences in repeat length3’. It is unlikely that the regional specificity of pathology is as a result of tissue variation in repeat lengths. The relative susceptibility of the striatum in HD is likely to be a function of some relatively unique feature of affected striatal neurons. A precedent exists in the form of another neurodegenerative disorder, familial amyotrophic lateral sclerosis (FALS). Some cases of FALS are caused by point mutations of the Zn2+/Cu2+ superoxide dismutase38 (SOD). While this enzyme is expressed widely throughout the body, only specific neuronal populations are affected in FALS. The gain of function

hypothesis

The function of huntingtin is currently unknown. Huntingtin lacks overall homology to previously characterized proteins but does possess the leucine zipper motif associated with gene regulatory proteins”. The CAG repeat would be translated as polyglutamine, a sequence that is found commonly in transcription factors39. Preliminary immunocytochemical localization of huntingtin suggests both cytoplasmic and nuclear localization16. A necessary step is the characterization of the function of huntingtin. The complete armamentarium of molecular and cell biology is being applied to this protein. Within the next couple of years there will be considerable data about the pattern of expression of huntingtin, its ultrastructural localization, regulation and function. The relatively high level of expression of IT15 mRNA within neurons suggests that this gene plays an important role in neuronal function, and characterization of huntingtin will undoubtedly yield very interesting results. There is reason to believe, however, that uncovering the normal function of huntingtin will not increase the understanding of the pathogenesis of neuronal death in HD. True dominant behavior has been suggested to result from so-called ‘gain of function’ mutations in which the affected locus acquires a new functional characteristic rather than loss of function of one allele. The major alternative is a ‘dominant negative’ effect in which an abnormality of one allele causes inactivation or dysfunction of both alleles. In the case of HD, the gain of function hypothesis is supported by several observations. Disruption or elimination of one IT15 allele does not cause HD. Individuals with the Wolf-Hirschhorn syndrome have deletions of the region of chromosome 4 encompassing IT15. While patients with WolfHirschhorn syndrome have a number of abnormalities, they do not exhibit HD-like neuropatholofl. Ambrose and colleagues have described recently an individual with a balanced translocation involving chromosome 4 in which the break occurred within the predicted open reading frame of IT15 (Ref. 15). This balanced translocation carrier is normal. Experience with another triplet-repeat disorder, SBMA, also supports the gain of function hypothesis’l. In SBMA, an X-linked disease, an excessive number of CAG repeats occurs within the coding region of the androgen-receptor gene. Affected males have a form of motor neuron disease, and often have mild androgen insensitivity. However, point mutations or deletions of the androgen receptor are described that

and D. Tagle

- Huntington’s

disease

YERsPECT~VE~ ONDlsEAsE

result in marked androgen insensitivity but not motor neuron disease. The implication is that loss of gene function does not cause motor neuron disease, and the expansion of the CAG repeat has resulted in the acquisition of a new property that causes motor neuron disease. Recent experiments with a transgenic mouse model of FALS also support the gain of function concept. Familial amyotrophic lateral sclerosis is dominantly inherited, and the effects of the SOD point mutation were thought originally to be a dominant negative effect ” . Transgenic animals that express the human mutant SOD possess normally functioning murine SOD, and develop degeneration of motor neurons, indicating that the human mutant SOD has acquired a new and lethal effectg3. Transgenic animals carrying a version of IT15 with expanded CAG repeats might provide an excellent animal model of HD. The gain of function hypothesis suggests that understanding the effects of excessive CAG repeats on gene function(s) will provide clues to the pathogenesis of neuron death in HD. Increased numbers of CAG repeats occur not only in HD and SBMA but also in SCAl (Ref. 44) and DRPLA (Refs 45-47). Understanding the effects of increased CAG repeats might increase our comprehension of mechanisms of neurodegeneration in a number of diseases. Excessive CAG repeats do not seem to affect the level of IT15 mRNA expression”, and most speculations about gain of function effects focus on changes in protein function. Possible

pathogenetic

mechanisms

Several interesting hypotheses have been proposed to account for the effects of excessive CAG repeats. Green has pointed out that polyglutamine stretches are potential substrates for transglutaminases which cause crosslinking of proteins and the formation of permanent glutamyl adductsg8. Potentially, the longer the polyglutamine stretch, the more efficient the crosslinking by transglutaminases. The transglutamination hypothesis can also account for the relatively later onset of HD by positing slow accumulation of abnormally transglutaminated proteins until some threshold is achieved beyond which neurons suffer fatal injury. Another interesting hypothesis that relates excessive numbers of CAG repeats to pathology is modulation of gene transcription39,41. The length of the polyglutamine tract influences the efficiency of transcription factors with a probable optimal length. Should huntingtin, either in its normal form or as a result of excessive CAG repeats, act as a transcription factor, it is easy to imagine how excessive CAG repeats could alter a number of cellular functions. Perutz and co-workers have articulated an intriguing variant of the gene-regulation hypothesisg9. These workers noted that polyglutamine stretches tend to aggregate together via strong hydrogen bonding. They speculate that excessive CAG repeats might cause huntingtin to act as a sink for transcription factors that possess polyglutamine stretches and, consequently, disrupt neuronal function. Alternatively, polyglutamine aggregation might simply result in insoluble and ultimately toxic precipitates within neurons. TIN.5 Vol. 18, No. 1, 1995

13

lJERs~ECT~~~s

0N

DISEASE

Yet

R. Albin

another

and D. Tank

variant

of

- Huntiwton's

the

disease

transcription

regulation

leagues” who noted that the associated with myotonic

by Wang and colexpanded CTG triplets dystrophy potentiate

stable

at the

hypothesis

has

been

nucleosome

proposed

formation

site

of

the

triplet

expansion. The generation of aberrant nucleosome positioning by expanded triplets could lead to blockade

of

transcription

or

alteration

of

local

chromatin

structure, or both. These types of effects could influence the expression of genes that contain the expanded trinucleotide repeat, and surrounding genes. It seems unlikely that this type of mechanism is operative in HD as HD homozygotes have been found largely to express normal levels of IT15 mRNA, and expression of IT15 mRNA from both normal

and

abnormal

alleles

in

heterozygotes

has

been documented14,“. Finally, Cha and Dure have suggested that huntingtin,

and

the

degenerative

equivalent disorders

proteins that

in

are

other

neuro-

characterized

by

excessive CAG repeats, might undergo normal proteolytic processing to produce polyglutaminecontaining polypeptides’l. The excessively large polyglutamine tracts might not be catabolized adequately. Polyamines have a multitude of interesting biological effects, including potentiation of NMDA-receptor activation and modulation of mitochondrial

function,

which

could

be

relevant

explains

the

in

this

context. Concluding None

remarks of

these

hypotheses

regional

selectivity of pathology in HD. Given the wide expression of IT15 mRNA, some relatively unique feature(s) of affected neurons must be important determinants of neuronal death in HD. Continuing investigation of the biology of the striatum might lead to the definition of characteristics among the subpopulations of striatal neurons that predispose some subpopulations to degeneration. When defined, knowledge of these characteristics might assist in understanding the mechanism(s) by which expanded CAG repeats lead to neuronal death. There is also no obvious connection between expanded CAG repeats and the excitotoxic mechanism speculated to be the proximate cause of neuronal death in HD. The reproduction of many features of HD striatal neuropathology by acute administration of NMDA-receptor agonists is strong evidence for an excitotoxic mechanism of neuronal death in HD. Reformulations of the excitotoxic hypothesis that stress impairment of cellular metabolic function, leading to enhanced neuronal susceptibility

to

excitotoxic

injury

(weak

or

indirect

excitotoxicity), provide a framework for reconciling a broad array of cellular disturbances with a Acknowledgements

We thank Sara Tallaksen-Greene, Terry Bazzett, Kirk Frey and Lany Elmer for helpful comments. Supported 4 grants NS19613and AGO8671 and the Hereditary Disease Foundation.

14

proximate

excitotoxic

death’0,52-54.

Future research on HD offers an oppor-

tunity

to

biology detailed

mechanism

integrate

and

genetics,

molecular

systems-level

understanding

of

of

biology,

neurobiology the

neuronal

pathogenesis

cell

into

a of

neurodegeneration. Selected 1 Folstein,

Families, 2 Quarrell,

(Harper,

references Disease: A Disorder of F’ress 0. and Harper, P. (1991) in Huntington’s Disease P.S., ed.), pp. 37-80, W.B. Saunders S.E.

Huntington’s Hopkins University

(1989)

The Johns

77h’S Vol. 18, No. 1, 1995

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