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Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Impaired remyelination and axonal degeneration may account for progressive disability in MS patients. As ciliary neurotrophic factor (CNTF) takes part in myelogenesis, we examined the frequency of a CNTF-null mutation in 349 MS patients with respect to their clinical presentation and in comparison with 434 healthy controls. Similar genotype frequencies for the CNTF mutation were obtained in MS patients (genotype 0101=74.8%, 0102=22.3%, 0202=2.9%) and controls (genotype 0101=71.7%, 0102=26.5%, 0202=1.8%) even after stratification for the HLA-DRB1*15 allele. In addition, there was no significant correlation of CNTF genotypes to age at onset, course or severity of the disease. We therefore conclude, that the requirement for CNTF in myelogenesis or cell survival may be bypassed by a second ligand or redundancy of functional activity of other neurotrophic factors.

Summary Background Narcolepsy is a common neuropsychiatric disorder characterized by increased daytime sleepiness, cataplexy and hypnagogic hallucinations. Deficiency of the hypocretin neurotransmitter system was shown to be involved in the pathogenesis of narcolepsy in animals and men. There are several hints that neurodegeneration of hypocretin producing neurons in the hypothalamus is the pathological correlate of narcolepsy. The ApoE4 allele is a major contributing factor to early-onset neuronal degeneration in Alzheimer disease and other neurodegenerative diseases as well. Methods To clarify whether the ApoE4 phenotype predisposes to narcolepsy or associates with an earlier disease onset, we have genotyped the ApoE gene in 103 patients with narcolepsy and 101 healthy controls. Results The frequency of the E4 allele of the ApoE gene was 11% in the patient and 15% in the control groups. Furthermore, the mean age of onset did not differ between the ApoE4+ and ApoE4- patient groups. Conclusion Our results exclude the ApoE4 allele as a major risk factor for narcolepsy.

The genetic basis of neurodegeneration in Huntington’s disease (HD) has been identified as a (CAG)>37 repeat expansion in a gene of unknown function. Interestingly, patients with the same expanded (CAG)n repeat length may have markedly different ages at onset. Based on experiences in animal models the level of expression might be one of the modifying factors. To gain insight into the regulation of the human HD gene we functionally analyzed 2266 bp of the HD gene promoter region. This region lacks a TATA and a CAAT box, is GCrich, and it has several consensus sequences for SP1, AP-2 and AP-4 binding sites. The stretch between nucleotides −49 and −198 relative to the first ATG is highly conserved between human and rodents and it harbors several potential binding sites for transcription factors. We analyzed deletion mutants fused with the chloramphenicol acetyltransferase (CAT) reporter gene in transfected, huntingtin expressing neuronal (NS20Y) and non-neuronal (CHO) cell lines. Partial deletion of the evolutionarily conserved part of the promoter significantly reduces the activity in both neuronal and non-neuronal cells indicating that the core promoter activity is located between nucleotides −221 and 4, relative to the +1 translation start site. Binding affinities of DNA–protein interactions were defined by electrophoretic mobility shift assays and the protected nucleotide positions were determined by DNase I footprinting.

Background: The authors identified the second known mutation in the alpha -synuclein (SNCA) gene, an alanine-to-proline exchange in amino acid position 30 (A30P), that cosegregates with the disease in one German family with autosomal dominantly inherited parkinsonism (ADP). The authors studied carriers of the A30P mutation to compare the phenotype of this mutation with idiopathic PD (:IPD) and to assess nigrostriatal dopaminergic function in symptomatic and preclinical mutation carriers. Methods: The pedigree of the A30P family spans five generations with five affected individuals. The authors performed detailed neurologic examinations followed by mutation analysis in 11 living individuals. In three mutation carriers, two individuals with definite PD and one person at risk for PD, they used L-[(18)]F-fluoro-3,4-dihydroxyphenylalanine (F-DOPA), [(11)]C-raclopride (RAC), and [(18)]F-fluorodeoxyglucose (FDG) PET to investigate presynaptic dopaminergic function, dopamine D, receptors, and cerebral energy metabolism. The authors studied the cognitive functions of carriers of the A30P mutation casing neuropsychological screening. Results: PET studies revealed striatal presynaptic dopaminergic alterations consistent with sporadic IPD in two affected family members and no evidence for nigrostriatal dopaminergic dysfunction in one presymptomatic mutation carrier. Neuropsychological testing in four mutation carriers provided evidence for cognitive impairment as a frequent and early symptom of the A30P mutation; this is also supported by regional cerebral energy metabolism alterations in the clinically presymptomatic subject. Conclusions: The phenotype of the A30P mutation in the SNCA gene is similar to that of sporadic IPD, including a high variability of the age at disease onset, ranging from 54 to 76 years. The follow-up of presymptomatic carriers of the A30P mutation may give insight into preclinical disease stages and early manifestations of PD.

Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD) are clinically overlapping syndromes, collectively called "peroxisome biogenesis disorders" (PBDs), with clinical features being most severe in ZS and least pronounced in IRD. Inheritance of these disorders is autosomal recessive. The peroxisome biogenesis disorders are genetically heterogeneous, having at least 12 different complementation groups (CGs). The gene affected in CG1 is PEX1. Approximately 65% of the patients with PBD harbor mutations in PEX1. In the present study, we used SSCP analysis to evaluate a series of patients belonging to CG1 for mutations in PEX1 and studied phenotype-genotype correlations. A complete lack of PEX1 protein was found to be associated with severe ZS; however, residual amounts of PEX1 protein were found in patients with the milder phenotypes, NALD and IRD. The majority of these latter patients carried at least one copy of the common G843D allele. When patient fibroblasts harboring this allele were grown at 30 degrees C, a two- to threefold increase in PEX1 protein levels was observed, associated with a recovery of peroxisomal function. This suggests that the G843D missense mutation results in a misfolded protein, which is more stable at lower temperatures. We conclude that the search for the factors and/or mechanisms that determine the stability of mutant PEX1 protein by high-throughput procedures will be a first step in the development of therapeutic strategies for patients with mild PBDs.

The principle of a novel typing techniques is described for screening with high efficiency the “immunological genome” by using various intronic and exonic oligonucleotide hybridization probes simultaneously or subsequently.

We investigated the gene encoding the β subunit of cGMP phosphodiesterase (PDE6B) as a candidate for generalized progressive retinal atrophy (gPRA), an autosomal recessively transmitted eye disease in dogs. The PDE6B gene was isolated from a genomic library. Single-strand conformation polymorphism analysis revealed eight intronic variations in different subsets of the 14 dog breeds investigated. In addition, we identified an 8-bp insertion after codon 816 in certain Sloughi dogs. Analysis of PRA-affected and obligatory carrier Sloughis showed that this mutation cosegregates with disease status in a large pedigree. All other exchanges identified were not located in functionally relevant parts of the gene (e.g., in the splice signal consensus sites). In most dog breeds (Labrador retriever, Tibetan mastiff, dachshund, Tibetan terrier, miniature poodle, Australian cattle dog, cocker spaniel, collie, Saarloos wolfhound, Chesapeake Bay retriever, and Yorkshire terrier), PDE6B was excluded as a candidate gene for gPRA because heterozygous allele constellations were detected in diseased animals. Therefore, the PDE6B sequence variations did not segregate together with the mutation(s) causing gPRA. Direct and indirect DNA tests concerning gPRA can be offered now for a variety of different dog breeds.

Immunomodulating factors have been shown to play a role in the pathogenesis of Parkinson's disease (PD) by biochemical methods. In order to investigate functionally important genes of the tumor necrosis factor alpha (TNFalpha) pathway we studied the frequency of DNA polymorphisms in the interleukin 6 (IL6), the TNFalpha, and the TNFalpha receptor 1 (TNFR1) genes in 264 sporadic German PD patients and in 183 age and sex matched German healthy controls. Analyzing the TNFalpha-308 polymorphism we found heterozygous individuals carrying alleles 1 and 2 more frequently in patients with a relative risk of 1.56 (p = 0.046, p(c) = 0.13, chi2 = 3.98). In contrast, the frequency of the B/2 haplotype described by the TNFR1-609 and TNFRI+36 polymorphisms was significantly decreased in our PD patients group (p = 0.0097, p(c) = 0.048, chi2 = 6.69) with a relative risk reduced to 0.52. Our results suggest an involvement of immunomodulating factors in the pathogenesis of sporadic PD as revealed by a molecular genetic approach.

To determine genetic susceptibility factors forHelicobacter pyloriinfection, polymorphic T-cell receptor gene elements were investigated in 203H. pylori-infected individuals and 180 uninfected individuals (controls).H. pyloriinfection is highly associated with individuals homozygous for the nonfunctional TCRBV6S1B element (odds ratio = 5.9; χ2= 13;P= 0.00032;Pvalue corrected for multiple comparisons [Bonferroni correction] = 0.00063).

A simple and fast method with high reliability is necessary for the identification of mutations, polymorphisms and sequence variants (MPSV) within many genes and many samples, e.g. for clarifying the genetic background of individuals with multifactorial diseases. Here we review our experience with the polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP) analysis to identify MPSV in a number of genes thought to be involved in the pathogenesis of multifactorial neurological disorders, including autoimmune diseases like multiple sclerosis (MS) and neurodegenerative disorders like Parkinsons disease (PD). The method is based on the property of the DNA that the electrophoretic mobility of single stranded nucleic acids depends not only on their size but also on their sequence. The target sequences were amplified, digested into fragments ranging from 50-240 base pairs (bp), heat-denatured and analysed on native polyacrylamide (PAA) gels of different composition. The analysis of a gre at number of different PCR products demonstrates that the detection rate of MPSV depends on the fragment lengths, the temperature during electrophoresis and the composition of the gel. In general, the detection of MPSV is neither influenced by their location within the DNA fragment nor by the type of substitution, i.e., transitions or transversions. The standard PCR/SSCP system described here provides high reliability and detection rates. It allows the efficient analysis of a large number of DNA samples and many different genes.

Summary Generalised progressive retinal atrophy (gPRA) is a heterogeneous group of hereditary diseases causing degeneration of the retina in dogs and cats. As a combination of mutations in theRDS/Peripherin and the ROM1 genes leads to the phenotype of retinitis pigmentosa in man we first performed mutation analysis to screen these genes for disease causing mutations followed by the investigation of a digenic inheritance in dogs. We cloned the RDS/Peripherin gene and investigated the RDS/Peripherin and ROM1 genes for disease causing mutations in 13 gPRA-affected dog breeds including healthy animals, obligate gPRA carriers and gPRA-affected dogs. We screened for mutations using single strand conformation polymorphism (SSCP) analysis. Sequence analysis revealed several sequence variations. In the coding region of the RDS/Peripherin gene three nucleotide exchanges were identified (A277C; C316T; G1255A), one of which leads to an amino acid substitution (Ala339Thr). Various silent sequence variations were found in the coding region of the ROM1 gene (A536G, G1006A, T1018C, T1111C, C1150T, C1195T), as well as an amino acid substitution (G252T; Ala54Ser). By excluding the respective gene as a cause for gPRA several sequence variations in the intronic regions were investigated. None of these sequence variations cosegregated with autosomal recessively (ar) transmitted gPRA in 11 breeds. The candidate geneRDS/Peripherin obviously does not harbour the critical mutation causing the autosomal recessive form of gPRA because diseased individuals show heterozygous genotypes for sequence variations in the Miniature Poodle, Dachshund, Australian Cattle Dog, Cocker Spaniel, Chesapeake Bay Retriever, Entlebucher Sennenhund, Sloughi, Yorkshire Terrier, Tibet Mastiff, Tibet Terrier and Labrador Retriever breeds. In the following breeds the ROM1 gene was also excluded indirectly for gPRA: Miniature Poodle, Dachshund, Australian Cattle Dog, Sloughi, Collie, Tibet Terrier, Labrador Retriever and Saarloos/Wolfhound. Digenic inheritance for gPRA is practically excluded for both these genes in four breeds: Miniature Poodle, Dachshund, Labrador Retriever and Saarloos/Wolfhound.

Friedreich ataxia occurs due to mutations in the gene encoding the mitochondrial protein frataxin. This (31)P magnetic resonance spectroscopy study on the calf muscle of Friedreich ataxia patients provides in vivo evidence of a severe impairment of mitochondrial function. Mitochondrial adenosine triphosphate resynthesis was studied by means of the post-exercise recovery of phosphocreatine. After ischemic exercise in calf muscles of all patients, phosphocreatine recovery was dramatically delayed. Time constants of recovery correlated with mutations of the frataxin gene, the age of the patients, and disease duration. (31)P magnetic resonance spectroscopy represents the first expedient tool for monitoring therapeutic trials in Friedreich ataxia non-invasively.

Simple DNA sequences can be used as practical tools for probing simple repeat elements. In 1986 we differentiated human genomes using the32P- labeled synthetic probes (GATA)4and (GACA)4and other sequentially sim-ple quadruplet repeat oligonucleatides [1]. Additional probes harboring sim-ple repeat motifs of from two to six nucleotides were subsequently sim-ple synthesized32P-labeled and hybridized to DNAs from various sources. In parallel, panels of 5’ biotinylated and/or digoxigenated oligonucleotides habe been chemically synthesized [14] and tested for multilocus DNA sim-ple in about 300 fungal, paint, and animal species [[3 13]. To date, in every species, at least one of the probes has been found to be informative with respect to genetic individualization[2].Using the probe (CAC)5or its complement (CTG)5, the demonstration of individuality is possible in every human being, except for monozygotic twins [9]. Nurnberg et al. [9] have also established the somatic stability of (CAC)5/(CTG)5fingerprints in sim-ple tissues and determined the mutation rate to be less than 0.001 per sim-ple per gamete. Somatic stability and similar or even reduced mutation rates have been determined for the (GACA)4fingerprint bands[11], which mainly stem from sequences in the nucleolus organizer regions (NOR) in humans [7].In comparison to other identification methods, the advantages of multilocus fingerprinting ingeneral include the technical simplicity and reproducibility, the high degree of informativeness, and, at the same time,absolute protection of personal data.

In allen untersuchten Eukaryoten inklusive der Spezies Homo sapiens sowie auch in vielen Prokaryoten kommen repetitive DNA-Sequenzen in teilweise sehr verschiedenen Organisationsformen vor (Epplen et al. 1997b). Dieses Phanomen der Redundanz in den modernen Genomen hat sich bisher gegenuber allen experimentellen Strategien und theoretischen Erklarungsversuchen als refraktar erwiesen. Auf der Basis gut bekannter evolutio- narer Ereignisse wie Gen- und Genomduplikation (Ohno 1970) ist die Entstehung mehrerer Genkopien oder gar ganzer Familien sinntragender DNA- Abschnitte ohne weiteres abzuleiten. Durch mehrere Genkopien (Redundanz) kann einerseits besser sichergestellt werden, das im komplexen System eines Organismus oder einer Zelle die lebensnotwendigen Proteine und damit Stoffwechselwege bzw. Strukturen auf jeden Fall zur Verfugung stehen. Selbst wenn einzelne Komponenten nicht einwandfrei funktionieren, schafft die Redundanz eine gesicherte Funktion (zur Theorie s. „ Automaton“ mit vielen redundanten Komponenten, de- ren Signale von einem Mehrheitsentscheidungsorgan analysiert und weitergeleitet werden; Neumann u. Burks 1966). Diese Erwagungen erscheinen gerade auch bei der inharenten Fehlerhaftigkeit biologischer Systeme plausibel. Andererseits stehen uberzahlige Kopien fur evolutionare Neuund Weiterentwicklungen zur Verfugung, da der Fortpflanzungserfolg (fitness) der Nachkommen eines Organismus und einer Art durch die Existenz einer einzelnen, einwandfrei funktionierenden Genkopie ausreichend abgesichert ist.

Wegener granulomatosis (WG), microscopic polyangiitis (MP), and Churg Strauss syndrome (CSS) are rare systemic autoimmune disorders. Common features are anti-neutrophil cytoplasmic antibodies (ANCA) in patient sera. Whereas WG patients show mainly anti-proteinase 3 ANCA, MP and CSS patients typically present anti-myeloperoxidase (MPO) ANCA. ANCA play an important role in the pathogenesis in the vessel wall by activating polymorphonuclear cells (PMN) and increased adhesivity between PMN and endothelial cells via adhesion molecules. Here we investigated major adhesion molecules as predisposition factors via common polymorphisms in or in the vicinity of the candidate genes ICAM-1, e-selectin, PLAUR, CD11b, and CD18. A restriction fragment-length polymorphism in exon 11 of the CD18 gene was associated with MPO-ANCA + systemic vasculitis. Our data indicate that a common variant of the CD18 gene confers increased risk for CSS and MP, supporting that genetic factors are involved in the etiology and pathogenesis of ANCA-associated systemic vasculitides.

The genetic aetiologies of at least 20% of autosomal dominant spinocerebellar ataxias (SCAs) have yet to be elucidated1. We have recently identified a novel form of autosomal dominant SCA, termed SCA12, in a large pedigree ('R') of German descent. The phenotype is variable, but the prototypical phenotype is that of a classic spinocerebellar ataxia, and the disease resembles the spinocerebellar ataxias more closely than any other form of neurodegenerative disorder. Age of onset ranges from 8 to 55 years. Most individuals present in the fourth decade with upper extremity tremor, progressing over several decades to include head tremor, gait ataxia, dysmetria, dysdiadokinesis, hyperreflexia, paucity of movement, abnormal eye movements and, in the oldest subjects, dementia. MRI or CT scans of five cases indicate both cortical and cerebellar atrophy.

Twin studies evidence that genetic factors of the host influence the acquisition and the clinical outcome of Helicobacter pylori infections in addition to bacterial and environmental factors. In the tumor necrosis factor (TNF) alpha-gene, allelic frequencies of the polymorphic microsatellite TNFa and the promoter polymorphism TNF-308 were studied for 209 H. pylori+ patients and compared to 184 H. pylori- controls. In the H. pylori+ group 34 individuals suffered from duodenal ulcer and 45 from gastric ulcer. Genotyping of the TNFa microsatellite and TNF-308 polymorphisms was performed after polymerase chain reaction by polyacrylamide gel electrophoresis (PAGE) and allele-specific oligonucleotide hybridizations, respectively. The phenotype frequency of microsatellite allele TNFa6 was lower in the H. pylori+ females as well as infected females with gastric ulcer compared to uninfected controls. Infected men with duodenal ulcer had a decreased frequency of allele TNFa10. The genotype TNF1/TNF1 of the polymorphism TNF-308 is a risk factor for duodenal ulcer in H. pylori+ females; p = 0.01; relative risk (RR) = 10.7; corrected p-value (Pc) = 0.05. Thus, the TNF region is crucial in the complex genetic predisposition for H. pylori infection for certain patient subgroups.

Neurophysiological and pharmacological studies suggest a major role of the GABAB receptor in the epileptogenesis of absence seizures. The gene encoding the human GABABR1 receptor (GABABR1) has recently been mapped to human chromosome 6p21.3 by in situ hybridization, a region that harbors a susceptibility locus (EJM1) for idiopathic generalized epilepsy (IGE). We investigated the hypothesis that the GABABR1 gene (GABBR1) represents a candidate gene for EJM1 by: (1) defining the precise localization approximately 130 kilobases telomeric to the HLA-F locus, (2) by characterizing its genomic organization, and (3) by mutation screening of the entire coding region of GABBR1 in 18 German patients with juvenile myoclonic epilepsy (JME) who were derived from families with evidence for linkage to chromosome 6p21.3 (cumulative lod score Z=3.17 at HLA-DQ). The GABAB receptor gene consists of 22 translated exons. The two alternative transcripts, GABABR1a and GABABR1b, are derived from the same locus but they differ in their alternative 5'-exons. Mutation analyses in JME revealed several DNA sequence polymorphisms, two of which result in amino acid changes occurring in all IGE-affected members of two families. However, clinically unaffected relatives did carry the same variations, excluding these amino acid substitutions as the cause for IGE in these families.

Juvenile myoclonic epilepsy (JME) is a genetically determined common subtype of idiopathic generalized epilepsy (IGE). Significant evidence for linkage has been reported for a susceptibility locus for JME in the chromosomal region 15q14 that harbors the gene encoding the alpha7 subunit of the neuronal nicotinic acetylcholine receptor (CHRNA7). The present study was designed to test the earlier linkage finding and to explore whether this susceptibility locus is involved in the epileptogenesis of a broader spectrum of IGE syndromes. Multipoint parametric and nonparametric linkage analyses with seven microsatellite polymorphisms encompassing the region of the CHRNA7 gene were performed using two diagnostic schemes of JME-related traits in two groups of multiplex families ascertained through probands with either JME (n = 27) or idiopathic absence epilepsy (n = 30). The present linkage study failed to replicate evidence for a major susceptibility locus for JME in the region encompassing the CHRNA7 gene. In addition, we found no hint in favor of linkage to 15q14 under the broad diagnostic scheme in any of the sets of families. If genetic variation in this region confers susceptibility to JME, then its effect size might be too small or its occurrence too rare to be detected in the investigated families.

The present association study tested whether length variations of two adjacent polymorphic CAG repeats in the coding sequence of a small-conductance, calcium-activated potassium channel (hKCa3) confer susceptibility to common subtypes of idiopathic generalized epilepsy (IGE). We found no significant difference in the allelic length distribution of the CAG repeats between 290 healthy German controls and the entire sample of 126 German IGE patients (Wilcoxon rank-sum test, P = 0.44) or two subgroups, comprising either 78 patients with juvenile myoclonic epilepsy (Wilcoxon rank-sum test, P = 0.74) or 59 patients with idiopathic absence epilepsies (Wilcoxon rank-sum test, P = 0.44). Moreover, the allelic distribution in parents-child trios of 46 IGE offspring did not differ significantly between the transmitted and non-transmitted parental alleles (Wilcoxon rank-sum test, P = 0.48). Therefore, our association study provides no evidence that length variations of polyglutamine arrays in the N-terminus of the hKCa3 channel exert a frequent and relevant effect in the epileptogenesis of common subtypes of IGE.

SNPs (single nucleotide polymorphism) are found approximately every 300–1000 bp within the human genome [1]. Point mutations can result in diseases if a conserved and functionally relevant nucleotide is affected as in many monofactorial disorders. On the other hand, polymorphisms, which by themselves do not cause a disease, may be relevant in combinations of certain alleles from different genes. The so-called genetic background is responsible for susceptiblity to infectious or autoimmune diseases. Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) involving T cells, B cells and macrophages as well as cytokines and interferons (IFNs) [2, 3]. Therefore polymorphisms in genes of the immunoregulatory network may influence the manifestation and course of the disease. Microsatellites within or close to candidate genes have been used to screen for genetic predisposition with considerable efficiency [4]. Yet not all candidate genes can be analysed by this approach.

1. DNA Fingerprinting of Prokaryotic Genomes.- 2. Plant DNA Fingerprinting and Profiling.- 3. DNA Fingerprinting and Profiling in Behavioural Ecology.- 4. DNA Profiling in Veterinary Medicine.- 5. Multilocus DNA Fingerprinting.- 6. Various Levels of (Epi)Genetic Diversities as Demonstrable via Simple Repeated Sequences.- 7. Forensics: Analysis of Short Tandem Repeat Loci by Multiplex PCR and Real Time Fluorescence Detection During Capillary Electrophoresis.- 8. Mitochondrial DNA: Diversity Analysis and Possible Pitfalls.- 9. PCR-based DNA Profiling of Human Y Chromosomes.- 10. The Use of Imperfect Microsatellites for DNA Fingerprinting and Population Genetics.- 11. PCR-SSCP: A Method for Reliably Detecting Single Nucleotide Polymorphisms in Multifactorial Diseases.- 12. Amplified Fragment Length Polymorphisms: A Non-Random PCR-Based Technique for Multilocus Sampling.- 13. Two-Dimensional DNA Fingerprinting.- 14. Statistical Approaches and Methods in Population Genetics Using Microsatellite Data.- 15. Statistical Inference from DNA Evidence.- Guide to Solutions.- Guide to Protocols.- Troubleshooting Guide.

Simple repetitive DNA is composed of short sequence motifs ranging from one to six bases in length reiterated tandemly for 5 to 100 times or more [1]. Such conspicuous blocks are known to be widely interspersed throughout eukaryotic genomes and in a number of prokaryotic chromosomes (see the chapter of van Helden, this volume). On the population level, longer simple repeat blocks tend to vary in the numbers of tandem repeat units, generating an enormous source of polymorphism. The mutation mechanism predominantly responsible for the vast extent of the polymorphy, the so-called repeat slippage [2] phenomenon, is not yet fully understood. Polymorphic simple repeats, i.e. microsatellites, are easily and rapidly typed by polymerase chain reaction (PCR). Semiautomatable typing of microsatellite alleles has revolutionized mapping of genomes and allowed construction of high-density genetic maps [3]. One consequence of these maps is that practically all monogenic diseases with known chromosomal localization can be diagnosed indirectly via linkage analysis in informative family situations [4]. But to date these efficient tools for genome research have remained essentially unfathomable in many aspects of biological functions and the meaning for disease development. Notwithstanding, we illustrate here how to use simple repeats for direct mutation diagnostics in a special class of human genetic diseases and outline the principles for individually typing genetic predispositions for (complex) diseases.

Autosomal dominant spinocerebellar ataxias (SCA) form a group of clinically and genetically heterogeneous neurodegenerative disorders. The defect responsible for SCA3/Machado‐Joseph disease (MJD) has been identified as an unstable and expanded (CAG)(n) trinucleotide repeat in the coding region of a novel gene of unknown function. The MJD1 gene product, ataxin‐3, exists in several isoforms. We generated polyclonal antisera against an alternate carboxy terminus of ataxin‐3. This isoform, ataxin‐3c, is expressed as a protein of approximately 42 kDa in normal individuals but is significantly enlarged in affected patients confirming that the CAG repeat is part of the ataxin‐3c isoform and is translated into a polyglutamine stretch, a feature common to all known CAG repeat disorders. Ataxin‐3 like immunoreactivity was observed in all human brain regions and peripheral organs studied. In neuronal cells of control individuals, ataxin‐3c was expressed cytoplasmatically and had a somatodendritic and axonal distribution. In SCA3 patients, however, C‐terminal ataxin‐3c antibodies as well as antiataxin‐3 monoclonal antibodies (1H9) and anti‐ubiquitin antibodies detected intranuclear inclusions (NIs) in neuronal cells of affected brain regions. A monoclonal antibody, 2B6, directed against an internal part of the protein, barely detected these NIs implying proteolytic cleavage of ataxin‐3 prior to its transport into the nucleus. These findings provide evidence that the alternate isoform of ataxin‐3 is involved in the pathogenesis of SCA3/MJD. Intranuclear protein aggregates appear as a common feature of neurodegenerative polyglutamine disorders.

Generalised progressive retinal atrophy (gPRA) is a heterogeneous group of hereditary diseases causing degeneration of the retina in dogs and other animals. The genetic origin is unknown in most cases. We have screened the coding sequence of the ROM1 gene for disease causing mutations in Tibetan Terriers, Miniature Poodles, Dachshunds and Chesapeake Bay Retrievers by single strand conformation polymorphism analysis (SSCP). Two polymorphisms have been identified by sequencing, one in exon 1 in all examined breeds (position 210: G-->A; Gly40Arg and position 252: G-->T; Ala53-Ser). Another polymorphism was present in exon 2 (position 1150: C-->T and position 1195: C-->T) segregating in Miniature Poodles. None of these polymorphisms were cosegregating with gPRA rendering a disease causing mutation in the ROM1 gene unlikely.

The properties of simple trinucleotide repeats generate increased interest as expansions of certain trinucleotide blocks cause human diseases. Here, we studied protein binding and structural features of a perfect (gaa.ttc)24 tract in its original genomic environment. Electrophoretic mobility shift assays revealed that HeLa nuclear proteins bind to the DNA fragment containing the (gaa.ttc)24 block. Competition experiments using simple (gt.ac)n repeats differing in length and flanking regions showed no cross-reactivity with the major retarded band. For the specific (gaa. ttc)n/protein complex, a binding constant of 9.3x10-9 mol/l was determined. DNase I footprinting revealed protein binding sites located exclusively within the repeat with a preference for the (gaa)24 strand. OsO4 and DEPC modifications followed by electrophoretic and electron microscopical analyses showed that the (gaa.ttc)24 block forms different types of intramolecular triple helices: Under superhelical stress, different H-DNA isomers are evident, whereas exclusively H-Y forms were detected in the relaxed state. Together, these data have functional implications for genomic (gaa.ttc)n tracts.

Summary: Purpose: The present replication study was designed to test the validity of a previously mapped susceptibility locus (EGI) for common subtypes of idiopathic generalized epilepsy (IGE) in chromosomal region 8q24. Methods: Thirty-eight multiplex families of probands with common IGE syndromes were included in the present study. Parametric and nonparametric multipoint linkage analyses were conducted between the IGE trait (either “idiopathic” generalized seizure or generalized spike-wave EEG discharges) and three microsatellite polymorphisms (D8S256, D8S284, 0881128) encompassing the putative EGI locus. Results: Parametric and nonparametric multipoint linkage analysis provided no evidence for linkage between the IGE trait and the markers encompassing the putative EGI locus. Moreover, we noted no indication favoring linkage to this chromosomal region in two distinct subsets of families subdivided by the absence (n = 18) or presence (n = 20) of family members with juvenile myoclonic epilepsy (JME). Conclusions: We failed to replicate evidence of a major locus (EGI) for common familial IGE in chromosome region 8q24. On the contrary, our present parametric linkage results provide evidence against linkage across the region under a broad range of genetic models. If there is a susceptibility locus for IGE in this region, the effect size or the proportion of linked families is too small to detect linkage in these families. Taking into account the problems in replicating initial linkage claims in oligogenic traits, further linkage studies in additional family sets are necessary to evaluate the validity of the previous linkage finding.

Purpose: Specific simple DNA repeats occur at the telomeric ends of mammalian chromosomes. Loss of (G+C)-rich repeats can result in genetic instability, associated with tumorigenesis. So far, data on telomere shortening have not been available for different types of soft-tissue tumors. Methods: Using tumor material and the blood of the corresponding patient, high-molecular-mass DNA was prepared by digestion with proteinase K and extraction with phenol/chloroform. A 10-μg sample of DNA was digested with the restriction enzyme HinfI. DNA fragments were separated in a 0.7% agarose gel, and in-gel hybridization was performed with the telomere-specific repeat probe (TTAGGG)3. Results: Shortening of the telomere repeat was observed in 14/30 soft-tissue tumors; 5 tumors showed elongated telomere repeats, whereas the telomeres appeared unchanged in 11 tumors. Decreased telomere repeat length correlated with advanced age, DNA ploidy, and a higher proliferation index. There was no association between telomere repeat length and tumor grade. Interestingly, in contrast to other entities, all malignant schwannomas and leiomyosarcomas showed significantly reduced telomere lengths. An explanation for the telomere heterogeneity in liposarcomas may include differential telomerase reactivation in well and poorly differentiated tumors. Conclusions: Telomere shortening is frequent but not a uniform phenomenon in different types of soft-tissue tumor. Studies on telomerase activity should be performed in the same cohort of sarcomas.

The purpose of this study was to screen for somatic changes in invasive breast tumors by multilocus DNA fingerprints comparing normal (blood) and malignant tissue samples from 34 patients. The comparison of lymph node–positive and node–negative breast carcinomas was of primary interest. After restriction enzyme digestion with HinfI and HaeIII, altered banding patterns were detected by using the oligonucleotide probe (GTG) 5 in 7 of 34 (20.5%) and in 3 of 34 (8.8%) tumors after hybridization with (GACA) 4 . The overall frequency of changes thus amounted to 29.4%. Because long (GACA) n repeat motifs, generating predominant DNA fingerprint bands, are localized on the short arms of the human acrocentric chromosomes, sequences that are important in breast carcinogenesis may be present in these regions. The overall methylation status of the DNA does not appear to be responsible for DNA fingerprint differences, as can be demonstrated with the restriction endonuclease HaeIII. DNA fingerprint differences did not correlate with tumor grade, stage, and hormone receptor status. Tumors with lymph-node metastases expressed DNA fingerprint differences more frequently.

Mutations in the gene encoding the alpha1A-calcium channel subunit play a causative role in the epileptogenesis of absence seizures in tottering mutant mice. The present family-based association and non-parametric linkage study tested the hypothesis that allelic variants of the homologous human gene (CACN1A4) confer susceptibility to common subtypes of idiopathic generalized epilepsy (IGE). An expressed polymorphic CAG trinucleotide repeat in the 3' end of the CACN1A4 gene was assessed in 70 families ascertained through members with either childhood (CAE) and juvenile absence epilepsy (JAE), or juvenile myoclonic epilepsy (JME). Our association analysis using the haplotype-based haplotype relative risk statistic provided no evidence for an allelic association of the CAG repeat polymorphism with either IGE, or CAE and JAE, or JME. We found no relation between the CAG repeat length and susceptibility neither to IGE, nor to CAE and JAE, nor to JME. Non-parametric linkage analysis revealed no evidence for linkage of IGE traits with the CACN1A4 locus in 42 families of patients with either CAE or JAE. A weak trend towards an excess of allele sharing (identity by descent) among family members affected by an IGE was obtained in 26 families of JME patients (Z[NPL] = 1.25 at theta = 0.000, p = 0.057). Taken together, we found no statistically significant evidence that genetic variants of the CACN1A4 gene play a causative role in the pathogenesis of common subtypes of IGE in humans.

OBJECTIVE Spinocerebellar ataxia type 6 (SCA6) is an autosomal dominant cerebellar ataxia (ADCA) of which the mutation causing the disease has recently been characterised as an expanded CAG trinucleotide repeat in the gene coding for the α 1A -subunit of the voltage dependent calcium channel. The aim was to further characterise the SCA6 phenotype METHODS The SCA6 mutation was investigated in 69 German families with ADCA and 61 patients with idiopathic sporadic cerebellar ataxia and the CAG repeat length of the expanded allele was correlated with the disease phenotype. RESULTS Expanded alleles were found in nine of 69 families as well as in four patients with sporadic disease. Disease onset ranged from 30 to 71 years of age and was significantly later than in other forms of ADCA. Age at onset correlated inversely with repeat length. The SCA6 phenotype comprises predominantly cerebellar signs in concordance with isolated cerebellar atrophy on MRI. Non-cerebellar systems were only mildly affected with external ophthalmoplegia, spasticity, peripheral neuropathy, and parkinsonism. Neither these clinical signs nor progression rate correlated with CAG repeat length. CONCLUSIONS This study provides the first detailed characterisation of the SCA6 phenotype. Clinical features apart from cerebellar signs were highly variable in patients with SCA6. By comparison with SCA1, SCA2, and SCA3 no clinical or electrophysiological finding was specific for SCA6. Therefore, the molecular defect cannot be predicted from clinical investigations. In Germany, SCA6 accounts for about 13% of families with ADCA. However, up to 30% of SCA6 kindreds may be misdiagnosed clinically as sporadic disease due to late manifestation in apparently healthy parents. Genetic testing is therefore recommended for the SCA6 mutation also in patients with putative sporadic ataxia.

We investigated whether the polymorphisms rs133946 and rs133945 in the promoter region of the synapsin III (SYN3) gene are associated with multiple sclerosis in German patients. Association of the SYN3 variations and haplotypes with MS is not evident in German MS patients - in contrast to a MS cohort from Italy, as reported previously.

Since the identification in 2004 of mutations within the leucine-rich repeat kinase 2 ( LRRK2 ) gene in patients with autosomal, dominantly inherited Parkinson’s disease, this gene has been the focus of research on Parkinson’s disease.1 Mutations in the LRRK2 gene and especially the common mutation G2019S may account for 1–2% of familial and 3–6% of sporadic cases of Parkinson’s disease, including those of early and late onset.2 The LRRK2 gene is situated on chromosome 12p11.2–q13.1 and encodes a large protein named dardarin. Dardarin contains several functional domains, including a leucine-rich repeat domain, WD40, renin–angiotensin system/guanosine triphosphatases and kinase domains. The presence of the leucine-rich repeat and WD40 domains suggests a role in protein–protein interaction. In addition, function was demonstrated kinase for dardarin in vitro.3 Some questions remain about the growing demand of DNA diagnostics and genetic counselling in Parkinson’s disease. Which subpopulation of patients with Parkinson’s disease harbours the highest risk of mutations in the LRRK2 gene? What is the frequency and penetrance of different LRRK2 mutations? Nine exons (exons 19, 24, 25, 29, 31, 34, 35, 38 and 41) of the LRRK2 gene were screened for sequence …

Background: Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant cerebellar ataxia (ADCA) for which the disease-causing mutation has recently been characterized as an expanded CAG trinucleotide repeat. We investigated 64 families of German ancestry with ADCA and 55 patients with sporadic ataxia for the SCA2 mutation. Results: Expanded alleles were found in 6 of the 64 families and in 1 patient with sporadic ataxia. This patient had a de novo mutation from an intermediate paternal allele. Length of repeats in 21 patients with SCA2 ranged from 36 to 52 CAG motifs and was inversely correlated with age at onset and progression of the disease. Expanded alleles were unstable during meiosis; paternal transmission especially caused significant anticipation of onset up to 26 years earlier. The SCA2 phenotype differed from those of SCA1 and SCA3 with higher frequencies of slowed ocular movements, postural and action tremor, myoclonus, and hyporeflexia. However, no single feature was sufficient to permit a specific clinical diagnosis. Conclusions: Spinocerebellar ataxia type 2 accounts for about 10% of German families with ADCA but may also be present in sporadic ataxia due to de novo mutations. Clinical features are highly variable among and even within families. However, the size of the expanded repeat influences the phenotype and is relevant for course and prognosis of the disease.

Simple repetitive DNA sequences are abundantly interspersed in eukaryote genomes and therefore useful in genome research and genetic fingerprinting in plants, fungi and animals, including man. Recently, simple repeats were also identified in some prokaryotic genomes. Hence the same probes can be applied for multilocus DNA fingerprinting in medically relevant bacteria. Simple repeats including composite dinucleotide microsatellites are differentially represented in different compartments of eukaryote genomes. Expanded triplet blocks in and around certain genes may, for example, cause so-called trinucleotide diseases in man. As a consequence, simple repetitive sequences should also be characterized with respect to their influences on the DNA structure, gene expression, genomic (in)stability and their development on an evolutionary time scale. Here three examples of microsatellites in the human major histocompatibility complex (HLA) are investigated, a (GT)n microsatellite situated 2 kb 5' off the lymphotoxin alpha (LTA) gene, a (GAA)n block in the 5' part of the HLA-F gene and a composite (GT)n(GA)m stretch in the second intron of HLA-DRBl genes. Grossly differing mutation rates are evident in these elements as well as varying linkage disequilibria. The unfolding of these simple repeats in distant human populations is covered including Caucasians, Bushmen and South American Indians. Furthermore, implications of simple repeat neighboring genes are discussed for the multifactorial diseases multiple sclerosis (MS), rheumatoid arthritis (RA) and early onset pauciarticular arthritis (EOPA). Polymorphisms of HLA-DRBl and T cell receptor beta variable (TCRBV) genes confer susceptibility for these autoimmune diseases as demonstrable by intronic simple repeat variability. Microsatellite polymorphisms within the TNF region reveal linkage disequilibria with HLA-DRBl and different promotor alleles of the TNFA gene. Disease associations with TNFA microsatellite alleles are, on the one hand, secondary to associations with HLA-DRBl genes (in MS) or they represent additional risk factors (in RA, EOPA) on the other hand. Evolutionary persistence, various structural conformations and the specific binding of nuclear proteins to several simple repeat sequences refute the preconceptions of biological insignificance for all of these ubiquitously interspersed elements.

Population genetic studies are mainly based on the description of genetic variability and on interpopulational comparisons using genetic distance measures. The evolutionary dynamics of the populations are inferred from these parameters and accurate estimates of gene flow may be critical. The present study reevaluates the role of gene flow in human populations by different statistical methods from a number of microsatellite and protein polymorphism data. The estimated number of individuals exchanged per generation (Nm) was greater than 1 in all data sets with all statistical methods. The correlation between geographic and genetic distances suggests a pattern of isolation by distance, characteristic of demographic and genetic equilibrium conditions among populations worldwide. Thus the high values of Nm may be interpreted as a reflection of high gene flow between geographically close populations. As expected, gene flow appears to exert a pivotal role in the genetic history of humans.

DNA duplications and deletions of a 1.5-Mb region in chromosome 17p11.2-12 comprising the gene encoding peripheral myelin protein 22 (PMP-22) are the common mutations in Charcot-Marie-Tooth disease type 1 (CMT1) and hereditary neuropathy with liability to pressure palsies (HNPP). A 1.7-kb recombination hotspot region has been identified within misaligned flanking repeats (CMT1-REP elements) by detection of CMT- and HNPP-specific junction fragments in Southern blot analyses. In order to simplify routine diagnosis we introduce a polymerase chain reaction-based method to identify directly specific REP junction fragments. Using this test, specific fragments were detected in approximately 67% of both CMT duplication and HNPP deletion cases. Polymorphism within a specific restriction enzyme recognition site is crucial for both Southern blot and PCR analyses of junction fragments.

Publisher Summary This chapter describes simple repetitive sequences in DNA databanks. A characteristic feature of all eukaryotic and most prokaryotic genomes is the presence of various amounts of different repetitive DNA sequences. An ever-increasing number of microsatellite loci has been identified inadvertently during sequencing specific genes in genomic DNA or isolated and developed specifically for various purposes. Several features of the loci such as Mendelian inheritance, high mutation rates and easy screening by protein catabolic rate make them powerful tools for population genetic studies. The critical point to reveal a realistic evolutionary picture of a species is to obtain appropriate genetic similarity measures. Mutations involving several repeat units have been inferred from multimodal distributions of allele frequencies. Modal peaks sometimes constitute allelic classes and no intermediate size alleles are observed between these classes invalidating the stepwise mutation assumption. Disease-causing trinucleotide blocks mutate up to sevenfold more often than tetranucleotide microsatellites. It is found that trinucleotide loci associated with diseases are under selective range constraints, introducing severe bias into the population genetic analysis.

Background: The bactericidal/permeability increasing protein (BPI) is involved in the elimination of gram-negative bacteria. A functionally relevant single nucleotide polymorphism of the BPI gene causes an amino acid exchange (Glu216Lys). Study: To evaluate whether this single nucleotide polymorphism contributes to the predisposition to inflammatory bowel disease, we compared the allele frequencies of 265 patients with Crohn's disease, 207 patients with ulcerative colitis, and 608 healthy controls. Results: The Glu/Glu genotype frequency was decreased significantly in Crohn's disease patients as compared with controls (P < 0.027). No differences were obvious in patients with ulcerative colitis. Conclusions: Failure of the innate intestinal immune system could be involved in the pathogenesis of Crohn's disease via reduced/impaired defense against gram-negative bacteria.

A complex network of genes determines sex in mammals. Here, we studied a European roe deer with an intersex phenotype that was consistent with a XY genotype with incomplete male-determination. Whole genome sequencing and quantitative real-time PCR analyses revealed a triple dose of the SOX9 gene, allowing insights into a new genetic defect in a wild animal.

strains isolated from dif- ferent geographic regions, we could detect no apparent differences on the microscopic level. Even using random amplified polymorphic DNA (RAPD) analysis we were not able to distinguish them (unpublished data). Poly- morphisms on the protein level have not yet been de- scribed for C.

The biological meaning of abundant simple repetitive DNA sequences in eukaryote genomes is obscure. Therefore, (GAA)n, (GT)n, and composite (GT)n(GA)m blocks were characterized for protein binding in the repeat and flanking sequences of cloned genomic DNA fragments. In gel mobility shift and competition assays the binding of nuclear proteins to the repeats was specific (including some flanking single copy sequences). DNase footprinting revealed the target sequences within and adjacent to the repeats. Chemical modifications (Os04, DAPC) DAmonstrated non-B DNA structures in the polypurine blocks. The binding of nuclear proteins in and around simple repeat sequences refute biological insignificance of all of these ubiquitously interspersed elements.

Ovine lymphocyte antigen is associated with reduced faecal egg counts in 4-month-old lambs following natural, predominantly Ostertagia circumcincta infection. International Journal for Parasitology 26: 423-428. Ten lymphocyte antigens were defined in a flock of Scottish Blackface sheep known to be naturally exposed to infection with Ostertagia circumcincta. Population and family studies suggested that the 10 antigens were products of class I loci. Antigen G13br was in linkage disequilibrium with allele g2 at the DRB1 locus. The g2 allele has previously been associated with reduced faecal egg counts in a different crop of lambs from the same farm. In this study antigen G13br was also associated with a reduction in faecal egg counts. The results provide partial confirmation of the role of the major histocompatibility complex in resistance to natural, predominantly O. circumcincta infection.

Schizophrenia is a psychiatric disorder associated with atypical handedness and language lateralization. However, the molecular mechanisms underlying these functional changes are still poorly understood. Therefore, the present study was aimed at investigating whether variation in schizophrenia-related genes modulates individual lateralization patterns. To this end, we genotyped 16 single nucleotide polymorphisms that have previously been linked to schizophrenia on a meta-analysis level in a sample of 444 genetically unrelated healthy participants and examined the association of these polymorphisms with handedness, footedness and language lateralization. We found a significant association of the cholecystokinin-A receptor \(\textit {(CCKAR)}\) gene variation rs1800857 and language lateralization assessed using the dichotic listening task. Individuals carrying the schizophrenia risk allele C of this polymorphism showed a marked reduction of the typical left-hemispheric dominance for language processing. Since the cholecystokinin A receptor is involved in dopamine release in the central nervous system, these findings suggest that genetic variation in this receptor may modulate language lateralization due to its impact on dopaminergic pathways.