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Advances in the genetics of movement disorders: implications for molecular diagnosis.

Recent developments in molecular genetics have had a profound influence on the diagnosis and classification of inherited movement disorders. Huntington's disease is caused by the expansion of an unstable trinucleotide repeat sequence. Molecular diagnosis can now be performed by a simple PCR-based assay, and the study of the effects of the repeat expansion on the function of the encoded protein will allow to elucidate the molecular pathogenesis of the disease. Wilson's disease is caused by a large number of different mutations, which complicates molecular diagnosis. Genes for a number of inherited dystonic syndromes have been mapped, one of them, the gene for dopa-responsive dystonia, has already been identified. The genetic basis of several other prevalent movement disorders, such as essential tremor and the restless-legs syndrome however, is still obscure. Current research is also directed at the identification of inherited risk-factors in genetically complex movement disorders, such as Parkinson's disease.

Atrophy↗

Ammonium transport in Escherichia coli: localization and nucleotide sequence of the amtA gene.

Escherichia coli expresses a concentrative ammonium (methylammonium) transport system which is strongly repressed under conditions of nitrogen excess. We have previously reported the cloning of a structural gene (amtA) for this transporter by complementation. In this study, a 3.4 kb HindIII-BamHI fragment containing amtA was cloned into the pBluescript KS(+) vector, and unidirectional nested deletions from each end of this 3.4 kb fragment were generated by exonuclease III digestion. The deletions were analysed by complementation of the structural gene mutation produced by Tn10 insertion. This allowed amtA to be localized within a 1.4 kb region which spans the site of the mutation. By application of the Sanger dideoxy method, we sequenced the region containing amtA. The gene contains an open reading frame which encodes a protein with a predicted molecular mass of 27 kDa. The open reading frame is preceded by a putative Shine-Dalgarno sequence and followed by an inverted repeat which might function as a simple transcription terminator. Hydropathic analysis of the inferred amino acid sequence of the gene product predicts that amtA encodes a cytoplasmic component of the ammonium transport system.

Amino Acid Sequence↗

The paradox of MHC-DRB exon/intron evolution: alpha-helix and beta-sheet encoding regions diverge while hypervariable intronic simple repeats coevolve with beta-sheet codons.

Twenty-one different caprine and 13 ovine MHC-DRB exon 2 sequences were determined including part of the adjacent introns containing simple repetitive (gt)n(ga)m elements. The positions for highly polymorphic DRB amino acids vary slightly among ungulates and other mammals. From man and mouse to ungulates the basic (gt)n(ga)m structure is fixed in evolution for 7 x 10(7) years whereas ample variations exist in the tandem (gt)n and (ga)m dinucleotides and especially their "degenerated" derivatives. Phylogenetic trees for the alpha-helices and beta-pleated sheets of the ungulate DRB sequences suggest different evolutionary histories. In hoofed animals as well as in humans DRB beta-sheet encoding sequences and adjacent intronic repeats can be assembled into virtually identical groups suggesting coevolution of noncoding as well as coding DNA. In contrast alpha-helices and C-terminal parts of the first DRB domain evolve distinctly. In the absence of a defined mechanism causing specific, site-directed mutations, double-recombination or gene-conversion-like events would readily explain this fact. The role of the intronic simple (gt)n(ga)m repeat is discussed with respect to these genetic exchange mechanisms during evolution.

Alleles↗

[Repeating sequences, organizing the telomeric region of chromosomes from the eukaryotic genome].

The telomeric regions of the eukaryotic genome are composed of different types of the repeated DNA sequences. Extended subtelomeric repeats (STR) are located close to the terminating repeats (TR) at the extreme ends of the chromosomes. The first part of the review is devoted to the TR sequences. All of them are the simple satellite-similar tandem repeats (T/A)1-8 G1-8. TR, as a substrate of specific telomere elongating enzyme telomerase, controls the whole replication of the chromosomal DNAs. Information accumulated up to this time is sufficient to ascertain the principal uniformity of the structure, the functions and organization of TR in all eukaryotic genomes. The second part of the review is devoted to the STR sequences. The STRs are prolonged repetitive sequences localized between the TR cluster and non-repetitive genes. The STRs have been identified in rye, yeast, Trypanosoma, Drosophila, Chironomus, murine and human genomes. It can be seen from the analysis of experimental results that the STRs of different organisms may have general properties. The information about the functions of STRs is now being obtained for only the STR of Drosophila. The STR is the complex repetitive family located both in telomeres and in heterochromatin. The sequences from the family were recently shown to be capable of translocation and curing destroyed chromosomal ends. According to the latest information, the STR sequences are organized in two different types of clusters in the Drosophila genome. The types have different organization and different genome localization. Is the organization of both telomeric and heterochromatin regions of the Drosophila genome from the same structural elements common for higher eukaryotes or it is the characteristic feature of the Drosophila genome only, is to be cleared.

Animals↗

Genome scanning by two-dimensional DNA typing: the use of repetitive DNA sequences for rapid mapping of genetic traits.

The existence of repetitive DNA sequences offers the possibility to assess the mammalian genome for individual variation in its entirety rather than at one or only a few sites. In order to fully explore the various sets of mammalian repeat sequences for this purpose, analytical tools are required which allow many if not all individual members of sets of repetitive elements to be resolved and identified in terms of location and allelic variation. We have applied and further developed an electrophoretic system, two-dimensional DNA typing, which may fulfill these requirements. The two-dimensional system combines separation of DNA fragments by size in a neutral gel, with separation by sequence composition in a denaturing gradient gel. By hybridization with minisatellite- and simple-sequence core probes and by inter-repeat polymerase chain reaction techniques, it is possible to obtain individual--and even chromosome-specific separation patterns that consist of hundreds of spots. Computerized image analysis and matching of such spot patterns allows the rapid assessment of multiple polymorphisms, spread over the genome, to monitor genetic variability in populations. When coupled to databases of polymorphic DNA markers with a known genomic location, two-dimensional DNA typing can greatly accelerate the mapping of genetic traits in humans, animals, and plants.

Alleles↗

Accurate, high-throughput typing of copy number variation using paralogue ratios from dispersed repeats.

Recent work has demonstrated an unexpected prevalence of copy number variation in the human genome, and has highlighted the part this variation may play in predisposition to common phenotypes. Some important genes vary in number over a high range (e.g. DEFB4, which commonly varies between two and seven copies), and have posed formidable technical challenges for accurate copy number typing, so that there are no simple, cheap, high-throughput approaches suitable for large-scale screening. We have developed a simple comparative PCR method based on dispersed repeat sequences, using a single pair of precisely designed primers to amplify products simultaneously from both test and reference loci, which are subsequently distinguished and quantified via internal sequence differences. We have validated the method for the measurement of copy number at DEFB4 by comparison of results from >800 DNA samples with copy number measurements by MAPH/REDVR, MLPA and array-CGH. The new Paralogue Ratio Test (PRT) method can require as little as 10 ng genomic DNA, appears to be comparable in accuracy to the other methods, and for the first time provides a rapid, simple and inexpensive method for copy number analysis, suitable for application to typing thousands of samples in large case-control association studies.

DNA↗

A mouse repeat sequence conserved in eukaryotic genomes.

To study the biological role of simple repetitive DNA sequences, we analysed a clone isolated from a mouse macrophage cDNA library called T2. This clone contains two simple repetitive sequences: a new sequence, 'CAGAGAGG', and a sequence previously described, 'GATA'. By sequencing analysis, an open reading frame, coding for 225 amino acids, is detected in the T2 clone. The new simple sequence is present thousands of time in the mouse genome, associated or not with the sequence 'GATA'. The sequence 'CAGAGAGG' is transcribed in messenger RNAs. Northern blots of RNAs extracted from adult tissues and from differentiated or non differentiated cell lines show a large number of transcripts that quantitatively decrease when in vitro differentiation occurs. Moreover, Southern blots of DNA extracted from different organisms, hybridize with a fragment containing only the 'CAGAGAGG' sequence, demonstrating that this sequence is represented in the genome of phylogenetically distant eukaryotes, and is highly conserved during eukaryotic evolution.

Amino Acid Sequence↗

Discrete-length repeated sequences in eukaryotic genomes.

Two of the four repeated DNA sequences near the 5' end of the silk fibroin gene hybridize with discrete-length families of repeated DNA. These two families comprise 0.5% of the animal's genome. A repeated sequence with a conserved length has also been found in the short class of moderately repeated sequences in the sea urchin. The discrete length, interspersion, and sequence fidelity of these moderately repeated sequences suggests that each has been multiplied as a discrete unit. Thus, transposition mechanisms may be responsible for the multiplication and dispersion of a large class of repeated sequences in phylogenetically diverse eukaryotic genomes. The repeat we have studied in most detail differs from previously described eukaryotic transposable elements: it is much shorter (1300 base pairs) and does not have terminal repetitions detectable by DNA hybridization. A simple technique for identifying such discrete-length repeated sequences is described.

Animals↗

Use of variable simple sequence motifs as genetic markers: application to study of myotonic dystrophy.

Among the many classes of repetitive elements present in the human genome, the ubiquitous "simple sequence motifs" (SSMs) composed of [A]n, [TG]n, [AG]n or codon-tandem repeats form a major source of genetic variation. Here we report a detailed molecular-genetic study of a "variable simple sequence motif" (VSSM) in the apolipoprotein C2 (apoC2) gene, which maps to the 19q13.2 region in the vicinity of the myotonic dystrophy (DM) locus. By combining in vitro DNA-amplification using the polymerase chain reaction and high-resolution gel electrophoresis, we could demonstrate a high degree of allelic variation with at least ten alleles, which differ in the number of repeated [TG] or [AG] dinucleotide units. Similar results were found for the somatostatin I gene locus. To evaluate the usefulness of SSM-length polymorphisms as genetic markers, the apoC2-VSSM was employed for linkage analysis in DM families. Our results establish that the orientation of the apolipoprotein gene cluster on 19q is cenapoE-apoC2-ter and indicate that the many thousands of structurally similar VSSMs in the human genome represent a rich source of highly informative genetic and diagnostic markers.

Apolipoprotein C-II↗

High polymorphism level of genomic sequences flanking insertion sites of human endogenous retroviral long terminal repeats.

The polymorphism at the multitude of loci adjacent to human endogenous retrovirus long terminal repeats (LTRs) was analyzed by a technique for whole genome differential display based on the PCR suppression effect that provides selective amplification and display of genomic sequences flanking interspersed repeated elements. This strategy is simple, target-specific, requires a small amount of DNA and provides reproducible and highly informative data. The average frequency of polymorphism observed in the vicinity of the LTR insertion sites was found to be about 12%. The high incidence of polymorphism within the LTR flanks together with the frequent location of LTRs near genes makes the LTR loci a useful source of polymorphic markers for gene mapping.

Chromosome Mapping↗

Circularization and cleavage of guinea pig cytomegalovirus genomes.

The mechanisms by which herpesvirus genome ends are fused to form circles after infection and are re-formed by cleavage from concatemeric DNA are unknown. We used the simple structure of guinea pig cytomegalovirus genomes, which have either one repeated DNA sequence at each end or one repeat at one end and no repeat at the other, to study these mechanisms. In circular DNA, two restriction fragments contained fused terminal sequences and had sizes consistent with the presence of single or double terminal repeats. This result implies a simple ligation of genomic ends and shows that circularization does not occur by annealing of single-stranded terminal repeats formed by exonuclease digestion. Cleavage to form the two genome types occurred at two sites, and homologies between these sites identified two potential cis elements that may be necessary for cleavage. One element coincided with the A-rich region of a pac2 sequence and had 9 of 11 bases identical between the two sites. The second element had six bases identical at both sites, in each case 7 bp from the termini. To confirm the presence of cis cleavage elements, a recombinant virus in which foreign sequences displaced the 6- and 11-bp elements 1 kb from the cleavage point was constructed. Cleavage at the disrupted site did not occur. In a second recombinant virus, restoration of 64 bases containing the 6- and 11-bp elements to the disrupted cleavage site restored cleavage. Therefore, cis cleavage elements exist within this 64-base region, and sequence conservation suggests that they are the 6- and 11-bp elements.

Animals↗

Motor representation of the hand in the human cortex: an f-MRI study with a conventional 1.5 T clinical unit.

The purpose of this study was to show a gradient of possible bilateral activation for movements of the non-dominant vs. dominant hand, as well as for areas involved in complex vs. simple hand movements. A standard 1.5 T magnetic resonance imaging (MRI) system has been utilized to localize the cortical motor hand areas, using the blood oxygen level dependent contrast (BOLDc) technique and single-section fast low-angle shot (FLASH) imaging. Ten normal right-handed subjects volunteered for the study. The motor tasks consisted of simple (flexion-extension) finger movements of either hand, and complex movements (finger-to-thumb opposition in a repeating, pre-planned sequence) of the non-dominant hand. Simple movements caused contralateral activation of the primary motor area (MA); ipsilateral activation was observed for the non-dominant hand only. Supplementary motor area (SMA) was also activated, with a clear contralateral prevalence. The ratio of bilateral activation of MA did not change with complex movements of the non-dominant hand, while SMA as well as lateral premotor area were largely bilaterally activated in this task. In conclusion, the ipsilateral MA is activated for movements--even simple--performed with the non-dominant hand. There is widespread functional activity, involving both contralateral and ipsilateral SMA, for complex movements.

Adult↗

Expansion of a (CAG)n repeat region in a sporadic case of HD.

The genetic mutation underlying Huntington's disease (HD) has been identified as an expansion and instability of a specific CAG repeat sequence in a gene on chromosome 4. A simple polymerase chain reaction assay has been used for the assessment of the (CAG)n expansion in a 72-year-old woman with typical HD symptoms, but no family history of the disorder. The DNA analysis showed that the patient had an allele with 41 repeat units, in the size range seen in HD chromosomes. Therefore, HD diagnosis is confirmed in this seemingly sporadic case and the disease is newly diagnosed in a large family. The risk of inheriting this unstable expanded allele is discussed. INTRODUCTION--The discovery of an expansion of a trinucleotide (CAG) repeat region in the IT15 gene on the short arm of chromosome 4 has identified the mutational mechanism causing Huntington's disease (HD) and enables the direct diagnosis of affected subjects based on DNA analysis alone. Here a 72-year-old woman with typical HD symptoms, but no family history of the disorder, has been unambiguously diagnosed by using a quick DNA analysis. This is relevant because the disease is newly diagnosed in a large family. MATERIAL AND METHODS--A labelled polymerase chain reaction (PCR) test has been used to amplify the repeat region of the IT15 gene and DNA fragments were analyzed by Polyacrylamide gel electrophoresis. RESULTS--The number the CAG repeats in the proband displayed two alleles of 23 and 41 repeats, respectively. Since normal chromosomes are reported to contain 11-34 repeats, the clinical appearance of HD in the proband is explained by the presence of the repeat expansion. DISCUSSION--The parents of the proposita both died aged over 80 y apparently without neurological signs referable to HD. Hence, this is presumably a sporadic case of the disease. Because of the length of 41 repeats of this HD chromosome, offspring of this proband could inherit the expanded allele with 37 repeats, as expected for the reversal of the trinucleotide expansion. A subject with this intermediate allele could be affected, but would not be affected if the HD IT gene with reduced triplets had recovered its normal function. Thus, in a seemingly sporadic case like the one reported here, despite the PCR analysis, the risk of transmission of HD to her offspring may remain uncertain.

Aged↗

Towards covering immunological genes with highly informative markers: a trans-species approach.

To establish a highly informative screening system for immunologically relevant genes ("immunoprinting") we co-amplified via polymerase chain reaction (PCR) polymorphic exons plus adjacent intronic simple repetitive dinucleotide stretches in the T-cell receptor (Tcr) Vb6 and Major Histocompatibility Complex (MHC)-DRB loci in man and several ungulate species. In both gene families the basic structure of the simple repeat was found to be preserved for more than 70 x 10(6) years in all investigated species. The simple repeats exhibit extensive length variability. Distinct exon sequences are correlated with a defined repeat length and substructure. In addition, PCR and the oligonucleotides for typing were applicable to a broad range of species from different mammalian orders. Multiplex PCR of different members of the Tcr Vb6 family and MHC-DRB resulted in a complex pattern similar to an oligolocus fingerprint. Hence immunoprinting can be employed for searching for associations of immunologically relevant genes with diseases even across species barriers.

Animals↗

Genes with triplet repeats: a new class of mutations causing neurological diseases.

Microsatellites, simple tamdem repeats of 2 to 4 nucleotide sequences, are widely distributed throughout the genome. Trinucleotide repeats are found every 300 to 500 kb. Recently, a new type of mutation was described involving a specific expansion of triplets within or in close proximity to a gene. Expanded triplets have been found in the genes causing six different neurological disorders: fragile X syndrome (FRAXA), spinal and bulbar muscular atrophy (SBMA), myotonic dystrophy (DM), Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA1), and dentato-rubra-pallidoluysian atrophy (DRPLA). These neurological disorders have in common a variable age of onset and clinical severity, as well as a decrease in the age of onset over generations, known as anticipation. These unusual characteristics are related to the observation that expanded repeats are unstable both in meioses and mitoses. A younger age of onset and an increase in severity correlate with a higher number of repeats. Interestingly, particular haplotypes are in disequilibrium with the mutation for FRAXA, DM and HD, suggesting instability for selected chromosomes. How expanded triplets affect the expression and the function of genes is still unknown. Since neurodegenerative disorders are often variable in age of onset and clinical severity, the list of expanding triplet mutations should increase in the very near future.

DNA↗

An improved microcomputer program for finding gene- or gene family-specific oligonucleotides suitable as primers for polymerase chain reactions or as probes.

We present here an easy-to-use computer program which finds oligonucleotides suitable as primers in polymerase chain reactions (PCR) or as probes for hybridization. In contrast to other programs used for this purpose, the additional advantage of this one is the possibility of directly detecting gene- as well as gene family-specific oligonucleotides. For this purpose, up to 200 different DNA sequences, of maximally 65,000 nucleotides each, can be scanned in a single search to ensure either single or multiple gene binding of the PCR primers or probes. Specific oligonucleotides for genes carrying internal repetitions and for single genes belonging to a set of highly conserved genes can also be detected. Many parameters such as exclusion of simple sequences, which are known to be highly repeated throughout various genomes or regions of stable secondary structures in both primer-primer and primer-template, can be taken into consideration and avoided. Furthermore, the G + C content and the length of the oligonucleotides can be changed in a broad range by the user.

Animals↗

Telomeres and telomerase.

Telomeres are the protective DNA-protein complexes found at the ends of eukaryotic chromosomes. Telomeric DNA consists of tandem repeats of a simple, often G-rich, sequence specified by the action of telomerase, and complete replication of telomeric DNA requires telomerase. Telomerase is a specialized cellular ribonucleoprotein reverse transcriptase. By copying a short template sequence within its intrinsic RNA moiety, telomerase synthesizes the telomeric DNA strand running 5' to 3' towards the distal end of the chromosome, thus extending it. Fusion of a telomere, either with another telomere or with a broken DNA end, generally constitutes a catastrophic event for genomic stability. Telomerase acts to prevent such fusions. The molecular consequences of telomere failure, and the molecular contributors to telomere function, with an emphasis on telomerase, are discussed here.

DNA Damage↗

Neurological proteins are not enriched for repetitive sequences.

Proteins associated with disease and development of the nervous system are thought to contain repetitive, simple sequences. However, genome-wide surveys for simple sequences within proteins have revealed that repetitive peptide sequences are the most frequent shared peptide segments among eukaryotic proteins, including those of Saccharomyces cerevisiae, which has few to no specialized developmental and neurological proteins. It is therefore of interest to determine if these specialized proteins have an excess of simple sequences when compared to other sets of compositionally similar proteins. We have determined the relative abundance of simple sequences within neurological proteins and find no excess of repetitive simple sequence within this class. In fact, polyglutamine repeats that are associated with many neurodegenerative diseases are no more abundant within neurological specialized proteins than within nonneurological collections of proteins. We also examined the codon composition of serine homopolymers to determine what forces may play a role in the evolution of extended homopolymers. Codon type homogeneity tends to be favored, suggesting replicative slippage instead of selection as the main force responsible for producing these homopolymers.

Alanine↗