Morbus Huntington – a human genetic model disease (2000)

Early on in his scientific career in human genetics, Ulrich Wolf was the first author to describe a deficiency on the short arms of a chromosome no. 4 (Wolf et al., 1965), comprising the deleted region responsible for causing a new syndromal entity, later depicted as the Wolf-Hirschhorn syndrome (Wolf et al., 1965; Hirschhorn et al., 1965). Being small but microscopically identifiable, this chromosome region also harbors the locus responsible for Huntington’s disease (HD; Gusella et al., 1983). The general meaning of the chromosome 4 deletion and especially the interpretation of this seminal observation is rated highly, given the unpretentious staining technology employed >35 years ago. Besides medical, biological questions were also to be answered and they outweigh methodological sophistication by far. Similarly, later on genetic sex determination was a prime challenge to U. Wolf’s scientific intellect. The small amount of extra genetic material in males vs. females amounts to little more than the interindividual variability in unrelated humans and it represents mostly genomic “desert” (Ohno and Yomo, 1991; Epplen et al., 1998), i.e. repetitious DNA sequences. In HD the critical difference appears as a little extra of a simple repetitive DNA stretch. The length of a perfectly repeated (CAG)n block exceeds a quite well defined threshold (The Huntington’s Disease Collaborative Research Group 1993, Laccone et al., 1999). This latter discovery paved the way for direct DNA tests and unprecedented diagnostic precision, predictive genetic counseling and basic biochemical research into the causal pathogenesis of HD as well as research in a number of disciplines relating to human genetics, including clinical sciences.(Simple) repetitive DNA is not yet fully understood (but see e.g. Britten and Davidson, 1971; Smith, 1976; Ohno, 1982). Since these repetitive elements are available as tools for solving biological or basic medical problems, they are used on a large scale in the form of microsatellites for genome mapping and whole genome screening projects. Up to 6000 microsatellites are combined nowadays, e.g. for identifying the genetic predisposition and protection factors in multiple sclerosis (see GAMES initiative [G enetic A nalysis of M ultiple sclerosis in E uropeanS]) after pooling of some 200 DNA samples each for patients and controls. In addition, indirect gene diagnostics in families is virtually completely dependent on these microsatellites (Epplen et al., 1997). It was a surprise that a particular form of microsatellite also plays a crucial role in human genetic diseases as demonstrated in HD and more than a dozen other trinucleotide expansion diseases (see Fig. 1; Cummings and Zoghbi, 2000). Based on the phenomenon of anticipation in HD families, the identification of HD as a (CAG)n block expansion disease has been less unexpected.Since 1993 in our diagnostic unit 1,109 tests were commissioned for purposes of differential diagnoses, whereas 362 persons at risk asked for this investigation. As expected for differential diagnostics the sex ratio of tested persons is practically equal (49% ♀♀). In contrast, nearly 58% of the persons at risk were women that passed through the complete procedure for predictive testing. 36% of the persons at risk were to be informed about expanded (CAG)n blocks, while the differential diagnosis HD was verified in 67% of the referrals. The distribution of the (CAG)n alleles in the Huntingtin gene from >3,000 chromosomes from patients and persons at risk under investigation for HD is depicted in Fig. 2a. The “gray range” of alleles with reduced penetrance represents only 0.32% of the alleles in our clientele. As expected, the distributions of the non-pathogenetic (CAG)n block lengths are quite similar in non-affected individuals and in Huntingtin mutation carriers. But there is a remarkable trend that carriers of expanded (CAG)n block lengths also share longer alleles on the non-pathogenic Huntingtin alleles: The frequencies of (CAG)n<18 lengths is higher in non-affected individuals whereas (CAG)n>17 alleles are more often observed on the second non-pathogenic chromosome of mutation carriers (Fig. 2b). – Quality management measures in DNA diagnostics for HD ensure high performance in many laboratories. Very rarely PCR artifacts are caused by variants in primer attachment sites (Williams et al., 2000), but they only very exceptionally affect the outcome and interpretation of the HD test because conventional Southern blot hybridizations as well as a number of different primer combinations are employed for PCRs with suspicious results.Given established DNA diagnostics and enormous amounts of data, (CAG)n expansion diseases and especially HD are also of interest with respect to genotype/phenotype relationships (Wolf, 1995). Since the principal molecular genetics is uniform and since the mechanisms of causal pathogenesis in the central nervous system are the same in HD (Turmaine et al., 2000), it is evident that the dependence of the phenotype on the genotype is poorly understood and remains quite complex (see below) due to the residual genetic background as well as elusive epigenetic phenomena. – Research on the genetics of HD has been teaching many a lesson and has posed even more surprising questions. How does Huntingtin protein influence the differentiation of hematologic stem cells? How does the accumulation of Huntingtin protein specifically abrogate the recognition of disgust (see below)? In this context some principal questions are to be asked – even though research in clinical and experimental psychology is advancing steadily: Should we refrain from studying genetic influences on human behavior? According to some basic discussions with U. Wolf in the seventies, especially rewarding could be studies concentrating on the genetic background of female/male conflicts.Like in the other few institutions concentrating on HD, it is our own clinical experience from the specialized ward for HD patients (12 beds) that there is high variability in the age of onset, clinical symptoms and course of the disease. During the last 5 years we have dealt with some 420 HD patients (54.5% ♀♀, 45.5% ♂♂) in this ward. 64% of patients ranged between 35 and 60 years of age (8–76 years). Neurological investigation comprises clinical examination and scoring of the patients according to the international validated Unified Huntington’s Disease Rating Scale (UHDRS), Total Functional Capacity Scale (TFS) and Independence Scale (IS) as well as apparative tests such as SSEP, CCT and special motor performance series.87% of the patients showed symptoms of chorea, 57% suffered from choreatic movements as the main clinical symptom. ∼20% of the patients presented with other movement disturbances like dystonia, tremor, myoclonus or akinesia. Akinesia as a leading sign was observed in ∼10%, mainly in patients with the akinetic-rigid Westphal variant of HD. Major depression as the main symptom was evident in ∼14%; 6% of the patients suffered from psychosis with illusions and hallucinations, 1.5% from obsessive-compulsive disorder. Only 1.4% of the patients presented with severe aggressive behaviour, a symptom often referred to be common in HD.Most of the patients with choreatic movement disorder are treated with antidopaminergic neuroleptics like tiaprid and tetrabenazine. A combination of both drugs administered with as low doses as possible shows optimal effects on hyperkinesia with a low rate of side effects. Patients with problems in swallowing after high-dose neuroleptics especially benefit from this medical regime. Dopaminergic drugs or even low-dose levodopa are administered in akinetic patients suffering from the Westphal-variant. Between 1995 and 1997 patients with major depression, especially those with sleep disturbances, were treated with classical antidepressant drugs like amitriptylin or more often with the atypical neuroleptic drug sulpirid. Recently reuptake inhibitors for serotonin are applied. Psychotic patients received highly potent neuroleptics and hypnotics during the acute phase and atypical neuroleptics and anxiolytic drugs for the treatment of the chronic phases. Besides medication patients are treated by logopedicians, occupational therapists and physiotherapists. Modern methods of nursing like basal stimulation are performed to either stimulate or sedate patients. These practices are demonstrated to other caregivers to facilitate home care.The clinical concept of the HZ NRW combines care for symptomatic patients in the different stages of the disease, counseling and advice for gene carriers and their families. The collaboration with the human genetic department of the HZ NRW includes crisis intervention in patients after disclosure of the DNA test result. Persons at risk as well as pre- or early symptomatic HD carriers, who ask for clinical diagnostics, are examined by using a battery of neurological and neuropsychological tests. In clinical routine we perform different subtests of the Aachener Aphasie Test, Multilingual Aphasia Examination, Wechsler Memory Scale, Rey-Osterrieth-Figur and Beck Depressions Inventar to address neuropsychological items like depression, dementia, vigilance, verbal and non-verbal declarative memory, non-motor conditional associative learning. Social interactions of HD patients are hampered. It has been reported e.g. that facial expressions of disgust are selectively not recognized by HD patients (Sprengelmeyer et al., 1996) and the responsible brain areas have later been identified (Sprengelmeyer et al., 1998). Intensified interdisciplinary research, especially in healthy HD mutation carriers may pave the way for new strategies in early diagnostics and therapy for HD, and it may help to understand behavioural peculiarities of the patients.At the onset of HD, psychiatric and cognitive impairment is common, but there are patients with very early symptoms of motor dysfunction as well (e.g. detected by investigation of error feedback control; Smith et al., 2000). The differences in the presentation of clinical features depend in part upon the sizes of the (CAG)n blocks. Patients with similar (CAG)n tract lengths, however, present with a high variability in clinical symptoms, especially in the age of onset with a range of 30–70 years in patients carrying (CAG)40–45 tracts (Duyao et al., 1993). On the other hand, despite the fact of anticipation, there are similarities in onset and course of the disease in members of the same family. Modifying factors for the clinical phenotype of the disease have to be examined in members of different generations in one family as well as in subjects of different origin but with equal trinucleotide block expansions. Exact physical and neuropsychological staging data have to be combined with the search for modifying genetic factors in the same patients. Recently, polymorphisms in the promoter region of the Huntingtin gene were also identified (Coles et al., 1998; Norremolle et al., 1999). Those variables as well as a number of Huntingtin-associated (HAP1, GAPD, Apopain and others) and interacting proteins (especially with the N-terminus of Huntingtin i.e. HYPA, HYPB, HYPC, N-CoR; Boutell et al., 1999) are of interest with respect to phenotypic expression.Neuropathological findings revealed new insights into the pathophysiology of HD. Protein aggregates containing fragments of Huntingtin were detected in nuclei and the cytoplasm of striatal and cortical neurons in HD patients (DiFiglia et al., 1997) underscoring the “toxic fragment hypothesis”. In fact, inhibition of the cleavage of Huntingtin protein in dominant-negative caspase-1 mutant transgenic mice delays the progression of the disease (Ona et al., 1999), thus opening new therapeutic strategies with caspase inhibitors like Minocycline (Chen et al., 2000). The role of these aggregates in the pathogenesis of HD is questionable, but the development of aggregation inhibitors will present additional therapeutic options (Heiser et al., 2000). Abrogation of gene expression in transgenic mouse models by using a tetracycline-regulatable system led to the disappearance of intracellular inclusions and amelioration of the behavioural phenotype (Yamamoto et al., 2000). This fact raises the possibility that symptoms in HD may not simply persist, but even remain reversible – at least in part, if there were a way to modify the expression of the Huntingtin gene in man.Prior pathophysiological studies in animals after injection of glutamate agonists showed similarities to HD and led to the theory of excitotoxicity (Olney, 1969; Beal et al., 1991). This rationale is supported by recent binding studies, which revealed changes in glutamate receptors due to disturbances in mRNA expression in transgenic mice (Cha et al., 1998). In the near future a clinical study with the glutamate-antagonist RiluzoleTM performed by the European Huntington’s Disease Initiative (EHDI) will reveal whether glutamate antagonism can positively influence the course of the disease. Changes in the oxidative metabolism and the occurrence of free radicals, triggered by an elevation of excitotoxic substances, may initiate cell death by increased oxidative stress (Beal, 1995). Antioxidative drugs like vitamin E and ubiquinone have been recommended to HD patients for a long time to improve brain metabolism. A significant delay in disease symptoms was recently revealed using creatine in transgenic mice (Ferrante et al., 2000). Taken together, transgenic mouse models opened new therapeutic strategies for HD patients and persons at risk. In accordance with this, clinical studies with HD patients have to be performed to verify the clinical relevance of respective theories. Given the HD patient numbers in specialized centers, these questions could and should be answered in due time.The demand for genetic counseling and gene diagnostics in families affected by HD did not decrease during the seven years following the description of the Huntingtin gene. Since 1993, more than 400 persons have asked for genetic counseling with reference to HD in our institution, most of them together with one or more relatives. While in many families the diagnosis of HD has been known for a long time (often for generations), clients in whose families the disease has been diagnosed just shortly before also ask for advice. More than 330 persons at-risk addressed us because they planned to undergo predictive gene testing for HD (Table 1), because they were thinking about this possibility or just because they needed more information as a basis for their decision. After one or more counseling sessions, 50% of these clients decided to refrain from the DNA test or they did not continue the pre-test counseling protocol – at least for several months. 166 at-risk persons took predictive DNA tests and were informed about the result. Differential diagnostic DNA tests in symptomatic patients were the reason for genetic counseling in a much lower number of The of diagnostic are referred to us from clinical institutions HZ NRW a counseling from the genetic a a and in some clinical are in the counseling This the general of human genetic e.g. it is on the with his with the to in his The for or the gene test not be or This does not to to or or the but which the is not or to the with different from his the first counseling general information about the disease, course and genetic therapeutic and diagnostic are given (see A family is established which not only information about and clinical course in affected but also often many insights into the and his the disease. The of with HD patients in their own families and their way of with the to influence their own of the disease risk and their to be or not to be to with a test (see also et al., 1999). The counseling is on the the for the test in the present of the The for a demand for the gene test can be quite the is the caused by the about the future disease which many at-risk persons to with than with a possible test result. to their dependent on the test while many of the at-risk individuals to the genetic risk for their and to to future HD, the to from therapeutic and with respect to are also as the reason for the DNA possible the or a part in this and following as it is of benefit for the to have a from his who during the whole counseling and after the disclosure of the test result. The of the gene test not only affect the persons at risk but in the same their and families and and et al., 1997). possible influences of the test on relationships are a major in pre-test counseling first in the genetic counseling unit is by a phase of for the this of the at-risk persons to from the test or to delay While studies to of the tested persons with to higher and more strategies et al., a direct of tested and persons revealed that both did not in these et al., 1997). who to on with the pre-test procedure a for have the to ask the for advice if there is and can have additional with the genetic The is not than after the first but many clients much longer to several to about their for or the HD gene several of have been reported et al., et al., et al., the to the test and his to with it is not for for the counseling care or even have been very in our In the to after disclosure of the test we have with the clients and clients who the information that they the HD gene to have neurological in the clinical ward in to or early symptoms of the disease. persons are this possibility also before or of the gene but in our experience they of it only only a but also a test has been most clients in the that they their for the test as are to with their than they did with even though there has been of an test e.g. changes in also test are rarely not reported to problems for the such as of with respect to affected family During the last several studies have examined the of the HD gene test of to one after disclosure of the test et al., et al., et al., only data are available on long effects of the gene test and et al., but such studies will be of interest in to improve the for the test for or the test especially not only the on the but also other individuals are affected by the result. 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Wolf more than 35 years to the genomic region of the Huntingtin gene. In many a respect research and patient care in HD may be as that of a human genetic disease. U. Wolf in human genetics the of which present us with in this is to Ulrich Wolf on the of his as from the of and at the for his way of advancing the of human On the other hand, U. Wolf’s in the of one than several of has yet to more in the of the human genome by a from NRW in