62 The species and chromosomal distribution of the centromeric -satellite I sequence from sheep in the tribe Caprini and other Bovidae Chaves R, Guedes-Pinto H, Heslop-Harrison JS, Schwarzacher T 67 TSPY variants in six loci on the human Y chromosome Dechend F, Williams G, Skawran B, Schubert S, Krawczak M, Tyler-Smith C, Schmidtke J 72 Local mechanisms in sex specific morphogenesis Drews U 81 Microdeletion 4p16.3 in three unrelated patients with Wolf-Hirschhorn syndrome Dufke A, Seidel J, Schöning M, Döbler-Neumann M, Kelbova C, Liehr T, Beensen V, Backsch C, Klein-Vogler U, Enders H 85 A second case of inv(4)pat with both recombinants in the offspring: rec dup(4q) in a girl with Wolf-Hirschhorn syndrome and rec dup(4p) Dufke A, Eggermann K, Balg S, Stengel-Rutkowski S, Enders H, Kaiser P

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 cases (∼50). The vast majority of diagnostic cases are referred to us from clinical institutions across Northrhine-Westfalia (NRW).The HZ NRW offers a multidisciplinary counseling approach. Apart from the genetic counselors, a psychologist, a social worker and in some cases clinical neurologists are involved in the counseling process. This process follows the general principals of human genetic counseling, e.g. it is focused on the client (together with his partner/his family), with the aim to support him in his individual decision-making. The decision for or against the gene test must not be influenced or guided. This strategy does not exclude to point to personal, familial or professional consequences or conflict situations (which the counselor recognizes, but which the client is not aware of) or to confront the counselee with views different from his own.During the first counseling session, general information about the disease, its variable course and genetic background, therapeutic approaches and diagnostic possibilities are given (see Table 2). A family tree is established which not only gives information about manifestation ages and clinical course in affected relatives, but also often allows many insights into the client's individual situation and his personal attitudes towards the disease. The clients' experiences (or lack of experiences) with HD patients in their own families and their families' way of dealing with the illness seem to strongly influence their own perception of the disease risk and their expectations to be able or not to be able to cope with a bad test result (see also Decruyenaere et al., 1999). The counseling process is focused on the question: Is the decision for the test right in the present situation of the client? The motivations for a client's demand for the gene test can be quite diverse. Among the predominating motivations is the immense psychological burden caused by the uncertainty about the future disease which many at-risk persons consider harder to deal with than with a possible unfavorable test result. Many young adults intend to take their reproductive decisions dependent on the test result, while many of the older at-risk individuals want to clear the genetic risk for their children and perhaps grandchildren. Plans to adjust one's living circumstances to future HD, the hope to profit from (future) therapeutic possibilities and concrete decisions with respect to professional career, financial planning etc. are also frequently quoted as the reason for the DNA test. Whenever possible the client's partner or a close friend takes part in this and all following conversations as it is of great benefit for the client to have a reliable companion from his personal surrounding who supports him during the whole counseling process and after the disclosure of the test result. The consequences of the gene test do not only affect the persons at risk themselves, but in the same measure their partners and families (Quaid and Wesson 1995; Sobel and Cowan 2000; Tibben et al., 1997). Therefore, possible influences of the test result on partner and/or intrafamilial relationships are a major topic in pre-test counseling sessions.The first appointment in the genetic counseling unit is followed by a phase of reflection for the client. At this stage (or later) ∼50% of the at-risk persons decide to withdraw from the test or to delay it. While earlier studies pointed to positive self-selection of the tested persons with regard to higher "ego strength" and more positive coping strategies (Decruyenaere et al., 1996), a direct comparison of tested and untested persons revealed that both groups did not differ significantly in these psychological characteristics (Decruyenaere et al., 1997). Clients who decide to go on with the pre-test procedure contact a psychologist for another in-depth conversation. They have the opportunity to ask the social worker for advice if there is need and can have additional conversations with the genetic counselor. The blood sample is not taken earlier than four weeks after the first consultation, but many clients wait much longer (up to several years) to grow certain about their decision for or against the HD gene test. Though several predictors of psychological adjustment have been reported (Codori et al., 1997; Decruyenaere et al., 1996; Dudok deWit et al., 1998b), the client's individual reaction to the test result and his ability to cope with it is not foreseeable, neither for himself nor for the counseling team. Critical situations making necessary close-meshed care or even hospital admission have been very rare in our experience. In the days to weeks after disclosure of the test result, we have regular telephone contacts with the clients and offer further meetings. Most clients who receive the information that they carry the HD gene decide to have specific neurological examinations in the clinical ward in order to exclude or eventually detect (and treat) early symptoms of the disease. At-risk persons are offered this possibility also before or independent of the gene test, but in our experience they make use of it only rarely. Not only when a negative (favorable), but also when a positive (unfavorable) test result has been reported, most clients point out in the follow-up contacts that they consider their decision for the test as right. They are able to cope with their novel situation better than they did with uncertainty previously, even though there has been negative impact of an unfavorable test result, e.g. changes in partner relationships. Notably, also favorable test results are rarely not reported to cause considerable problems for the client, such as feelings of guilt with respect to affected family members. During the last years, several studies have examined the psychosocial consequences of the HD gene test within periods of six months to one year after disclosure of the test result (Codori et al., 1997; Decruyenaere et al., 1996; Dudok deWit et al., 1998a, b). So far, only limited data are available on long term effects of the gene test (Taylor and Myers, 1997; Tibben et al., 1997), but such long-term follow-up studies will be of great interest in order to further improve the support for the test participants.The decision for or against the test becomes especially problematic when not only the person deciding on the test, but also other individuals are affected by the result. This is the case, when 25% at-risk individuals ask for direct gene diagnostics and they have an asymptomatic 50% at-risk parent who does not wish to clarify his/her genetic status. The test does not only disclose the genetic status of the tested person, but it can also change the genetic risk of the parent and siblings. In this situation it is the aim of the counseling process to find a solution that is acceptable to all family members, if possible in conversations in which all person(s) affected by the test result take part (see Maat-Kievit et al., 1999).An even more complex situation arises when prenatal diagnostics is concerned. In our institution genetic counseling with respect to HD has been made use of so far by 23 families with existing pregnancies (Table 3). Though rather small in number, these counseling situations are always extremely demanding, both, in time requirements as well as from the emotional impact. The individual situations of the pregnant women, their partners and families are very different:– Was the genetic risk/carrier status known before the pregnancy?– Is the pregnancy planned/wanted?– Are there additional problems except for the HD risk?– What is the woman's/couple's attitude towards prenatal diagnostics etc.?All these counseling cases have in common that the limiting time is of major importance: no time is left to wait until the client feels ready to take the far-reaching decision of having the predictive HD gene test or even to make use of prenatal diagnostics.Very occasionally, gene diagnostics in predictive HD testing discloses results that are difficult to interpret with respect to the client's disease risk, e.g. HD alleles in the intermediate length zone of the (CAG)n block or homozygosity for the HD allele. In order to provide a specific risk assessment to these clients, it becomes necessary to take into account pedigree information and – if possible and wanted by the family – to include other family members in combined genetic and neurologic examinations. These examples underline the importance of close interaction between the genetic and clinical part of the HZ NRW.Predictive gene diagnostics has become possible in a growing number of diseases amongst which are familial cancer syndromes, other late manifesting neurogenetic diseases etc. In genetic counseling concerning these diseases, the experiences with HD turn out to be of great value. Psychological consequences of genetic tests for different diseases and the course of distress experienced by persons at risk during the respective counseling program show remarkable parallels (Dudok deWit et al., 1998a, 1998b). Despite disease specific differences, the general approach used in counseling HD families can be applied in other counseling situations. On the basis of interdisciplinary care giving (by human geneticists, clinicians, psychologists, social workers etc.), the client is supported to assess the far-reaching implications of the DNA test for himself/his family and to consciously decide if he wants to gain knowledge about his genetic status. In conclusion, not only on the level of molecular pathogenesis, but also in the practical application of scientific results, HD can be regarded as a human genetic model disease.The scope of this article is to illustrate some aspects of HD research, progress in patient care as well as local developments in the HZ NRW. A seminal discovery of U. Wolf more than 35 years ago relates directly to the genomic region of the Huntingtin gene. In many a respect research and patient care in HD may be regarded as that of a human genetic model disease. By subtly provoking young colleagues, diligently, U. Wolf planted hidden seeds in human genetics the blossoms of which present us with pleasant surprises in this widespread field.This article is dedicated to Professor Dr. rer. nat. Ulrich Wolf on the occasion of his retirement as emeritus from the chair of Human Genetics and Anthropology at the Albert-Ludwigs Universität Freiburg, Germany. We thank him for his discrete way of advancing the subject of modern human genetics. On the other hand, U. Wolf's working ethos expressed in the sense of "rather plan one truly elegant experiment than do several of them" has yet to become more widely perceived in the era of the human genome project.Supported by a grant from MSWWF NRW in 1993.

impala) 62 lervia (aoudad) 62 Aotus trivirgatus (New World monkey) 113 Bos taurus (bovine) 62, 113 Canis familiaris (dog) 113 Capra hircus (goat) 62 Capreolus capreolus (roe) 113 Connochaetes taurinus (gnu) 62 Equus caballus (horse) 113 Hippotragus niger (sable antelope) 62 Kobus ellipsiprymnus, K. kob, K. leche, K. megaceros (antelope) 128 Macaca fascicularis (Old World monkey) 113