New problems in testing for Huntington's disease: the issue of intermediate and reduced penetrance alleles.
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Alleles↗
Biomedical subjects
G J Van Ommen
Publications and source records attributed to G J Van Ommen.
The protein truncation test: A review.
Only changes in the DNA sequence manifesting deleterious effects at a functional level provide "disease-causing" mutations. Consequently, mutation-scanning techniques applied on a protein level would be most informative. However, because of a lack of functional knowledge and powerful methods, most currently applied techniques try to resolve mutations at the DNA level. The protein truncation test (PTT) provides a rare exception, targeting mutations that generate shortened proteins, mainly premature translation termination. PTT has several attractive characteristics, including pinpointing the site of a mutation, good sensitivity, a low false-positive rate, and, more importantly, the near-exclusive highlighting of disease-causing mutations. In addition, PTT facilitated the detection of a new mutation type, i.e., a sequence change generating a hypermutable region surfacing in the RNA. The main technical problems are related to the fact that PTT generally uses an RNA target, including the difficulties that arise from the potential differential expression and stability of the transcripts derived from the two alleles present. The PTT has hardly evolved from the method originally described, with multiplexing and N-terminal protein tagging forming the only innovating modifications. To implement high-throughput screens using PTT, major improvements of the basic procedure will be required.
Base Sequence↗
Distribution of inclusions in neuronal nuclei and dystrophic neurites in Huntington disease brain.
Recently, an N-terminal fragment of huntingtin was localized to neuronal intranuclear inclusions (NII), presumed to cause cellular dysfunction, and to inclusions in dystrophic neurites (IDN) in the neostriatum and neocortex of Huntington disease (HD) patients. In the present immunohistochemical study of autopsy brain of 2 juvenile-onset HD patients, 5 HD patients with adult-onset, and 5 controls, NII and IDN as stained with both N-terminal antiserum to huntingtin and ubiquitin antiserum were detected in the HD neostriatum, neocortex, and allocortex, but not in the HD pallidum, cerebellum, and substantia nigra nor in control brain. The frequency of NII in the HD neocortex was highest in the juvenile patients. Within the allocortex, NII and IDN were found in the entorhinal region, subiculum, and pyramidal cell layer of Ammon's horn. N-terminal huntingtin antiserum also labeled intranuclear granular structures adjacent to the neuronal nuclear membrane in 5 HD patients, one control with idiopathic epilepsy, and one with Alzheimer disease. Our results show that NII formation in HD involves the allocortex in addition to the neostriatum and neocortex. The development of NII in the neocortex and allocortex in HD brain might contribute to the emergence of the cognitive and behavioral symptoms of the disease.
Adolescent↗
Two-colour immunocytochemical staining of gamma (gamma) and epsilon (epsilon) type haemoglobin in fetal red cells.
We have developed a two-colour immunocytochemical staining method for the detection of fetal and embryonic haemoglobin in erythroid cells. The method was applied to study these haemoglobin types in fetal red cells. Specimens from fetal blood (10 weeks), cord blood and fetal liver (14 weeks) as well as chorionic villus samples (10-13 weeks) were stained for gamma and epsilon chains using CY3 and FITC labelled antibodies. Morphometric analysis was applied to determine cell size. Samples from organs involved in early embryonic development contained relatively large erythroblasts expressing the epsilon globin chain (megaloblasts); later in gestation the gamma chain was co-expressed by the same cells which ultimately became smaller and contained HbF (alpha 2 gamma 2) only. This phenomenon was confirmed in CVS samples in which all cell types were abundantly present. Since fetal erythroblasts are considered candidate cells for non-invasive prenatal diagnosis using FISH, we studied the phenotype of erythroblasts circulating in the maternal blood. The majority of erythroblasts in maternal blood appeared to be of the relatively small gamma globin-containing cell type. However, careful screening of the same maternal blood samples also revealed erythroblasts expressing epsilon or epsilon and gamma globins simultaneously, although at low frequency. Control specimens from non-pregnant women did not show nucleated red cells expressing either of the haemoglobin types. These observations may contribute to the better recognition of fetal cells in the maternal blood for prenatal diagnosis.
Chorionic Villi Sampling↗
Clinical diagnosis of heterozygous dystrophin gene deletions by fluorescence in situ hybridization.
Two-thirds of patients affected by Duchenne or Becker muscular dystrophy (DMD/BMD) carry large intra-genic deletions in the dystrophin gene. In males, the deletions can be efficiently detected using multiplex polymerase chain reaction (PCR) and Southern blotting. In contrast, deletion detection in carrier females is complicated by the presence of a normal gene copy on the second X-chromosome. We have analyzed the boundaries of 570 deletions and 34 duplications in the dystrophin gene identified in the São Paulo and Leiden diagnostic laboratories. The data were used to select an optimal set of cosmid probes for the detection of the most frequently deleted areas of the dystrophin gene. Six cosmids were evaluated in fluorescence in situ hybridization (FISH) experiments to assess deletions in 21 heterozygous deletion-carriers and nine controls. No discrepancy was found between the FISH analysis and the molecular data, demonstrating the accuracy of the technique for carrier detection in Duchenne and Becker muscular dystrophy.
DNA Probes↗
Prenatal diagnosis of trisomy 13 on fetal cells obtained from maternal blood after minor enrichment.
In a pilot study to establish fetal nucleated red blood cell (NRBC) detection in maternal blood, trisomy 13 was diagnosed by FISH analysis at 11 weeks' gestation. The NRBCs were detected after a single-step ficoll density gradient enrichment. In blood samples taken both before and after CVS, 52 and 80 NRBCs, respectively, were found to be positive for fetal haemoglobin. In 47 per cent of these cells, FISH analysis for X and Y chromosomes confirmed the fetal sex. Moreover, 48 per cent of these NRBCs showed three fluorescent signals for a chromosome 13 probe, which confirmed the diagnosis of trisomy 13, previously detected at CVS karyotyping. This is the first report of non-invasive prenatal diagnosis of trisomy 13, i.e., pre-CVS, in the first trimester. The high number of fetal NRBCs detected indicates a connection with aneuploidy, probably due to early impairment of the feto-maternal barrier.
Adult↗
High-resolution mapping by YAC fragmentation of a 2.5-Mb Xp22 region containing the human RS, KFSD and CLS disease genes.
The disease loci for X-linked Retinoschisis (RS), Keratosis follicularis spinulosa decalvans (KFSD), and Coffin-Lowry syndrome (CLS) have been localized to the same, small region in Xp22 on the human X Chromosome (Chr). To generate a high-resolution map of the available contig in this area, we have used the YAC fragmentation vectors pBP108/ADE2 and pBP109/ADE2 and generated fragmented YACs from a 2.5-Mb YAC (y939H7) spanning the mentioned disease gene candidate regions. Forty-seven fragmented YACs were generated and analyzed, ranging in size from 170 kb to over 2400 kb. The resulting YAC fragmentation panel was used to construct a detailed restriction map of the region and has been used to bin clones and markers. As a deletion panel, it will present a valuable resource for further mapping.
Abnormalities, Multiple↗
Pitt-Rogers-Danks syndrome and Wolf-Hirschhorn syndrome are caused by a deletion in the same region on chromosome 4p 16.3.
Recently, a deletion of chromosome 4pter was found in three patients with Pitt-Rogers-Danks syndrome. We investigated two of these patients, by means of DNA and FISH studies, together with two additional patients with Pitt-Rogers-Danks syndrome, to determine the critical region of the deletion in these patients and to compare this with the critical region in Wolf-Hirschhorn syndrome. All four patients showed terminal deletions of chromosome 4p of different sizes. One of them appeared to have an unbalanced karyotype caused by a cryptic translocation t(4;8) in the mother, resulting in a deletion of chromosome 4pter and a duplication of chromosome 8pter. The localisation of the Wolf-Hirschhorn critical region has been confined to approximately 1 Mb between D4S43 and D4S115. Our study shows that the deletions in four patients with the Pitt-Rogers-Danks syndrome overlap the Wolf-Hirschhorn critical region and extend beyond this in both directions. This study, combined with the fact that our third patient, who was previously described as a Pitt-Rogers-Danks patient, but who now more closely resembles a Wolf-Hirschhorn patient, makes it likely that Pitt-Rogers-Danks and Wolf-Hirschhorn syndromes are different clinical phenotypes resulting from a deletion in the same microscopic region on chromosome 4p16.
Abnormalities, Multiple↗
Subcellular localization of the Huntington's disease gene product in cell lines by immunofluorescence and biochemical subcellular fractionation.
Huntington's disease is a progressive neurodegenerative disorder, which is caused by expansion of a polymorphic (CAG)n repeat in the coding region of the Huntington's disease gene. The function of huntingtin has not been elucidated so far. Accordingly, detailed subcellular localization studies remain useful. In an immunohistochemical study, we have reported huntingtin to be present in the cytoplasm of cells in the majority of the tissues studied. In addition, we detected a signal in the nucleus of cells in some tissues, including neuronal cells. We have further extended these studies in various mammalian cell lines, using a panel of (affinity-purified) polyclonal huntingtin antibodies in immunofluorescence, confocal laser scanning microscopy and biochemical subcellular fractionation studies. In mouse embryonic fibroblasts, human skin fibroblasts and in mouse neuroblastoma cells huntingtin was present in the cytoplasm. All five antibodies, directed against different parts of huntingtin, also showed a signal in the nucleus. This signal could be competed by the original antigen. The localization of huntingtin in both cytoplasm and nucleus, was confirmed by biochemical subcellular fractionation studies. However, in most other studies, a nuclear location for huntingtin has not been found. Our results suggest, however, that besides its function(s) in the cytoplasm, a nuclear function of huntingtin at some stages of differentiation or in some phases of the cell cycle may not be excluded.
Animals↗
Somatic expansion of the (CAG)n repeat in Huntington disease brains.
The mutation causing Huntington disease (HD) has been identified as an expansion of a polymorphic (CAG)n repeat in the 5' part of the huntingtin gene. The specific neuropathology of HD, viz. selective neuronal loss in the caudate nucleus and putamen, cannot be explained by the widespread expression of the gene. Since somatic expansion is observed in affected tissue in myotonic dystrophy, we have studied the length of the (CAG)n repeat in various regions of the brain. Although we have not found clear differences when comparing severely and mildly affected regions, we have observed a minor increase in repeat length upon comparison of affected brain samples with cerebellum or peripheral blood. Hence, although further somatic amplification seems to occur in affected areas of the brain, the differences between affected and unaffected regions are too small to make this mechanism an obvious candidate for the cause of differential neuronal degeneration in HD.
Aged↗
High resolution DNA fiber-fish on yeast artificial chromosomes: direct visualization of DNA replication.
Fluorescent in situ hybridization (FISH) is a powerful, direct and sensitive technique with a wide resolution range that enables the simultaneous study of multiple targets, labelled in different colours. Spreading techniques, denoted here as 'Fiber-FISH', increase FISH-resolution to the DNA fiber, using decondensed nuclear DNA as hybridization target. FISH could be a powerful analytical tool for thorough physical examination of yeast artificial chromosomes (YACs) which are often chimaeric or contain internal deletions. However, with one exception restricted to meiotic yeast chromosomes, FISH has not been used successfully on yeast/YAC DNA. We have developed a fast and simple method that can be applied routinely for compositional and structural analysis of cosmid and YAC DNA in yeast. It enables precise localization and ordering of clones, resolves overlaps and distances and gives a detailed picture of the integrity and colinearity of both probe and target. The combination of high resolution, signal abundance and short yeast cell cycle allows direct visualization of replicating DNA fibers. In a 400 kb region of the human dystrophin gene, we identified two replication origins, demonstrating that human DNA cloned in yeast is capable of initiating its own replication.
Chromosomes, Artificial, Yeast↗
Subregional localization of 14 yeast artificial chromosomes to human chromosome region 1p by fluorescence in situ hybridization.
We have sublocalized to the region between 1p22 and 1p33 a total of 14 yeast artificial chromosomes previously assigned to a broader area of human chromosome 1p. Our purpose was to map DNA sequences that could be used for the molecular characterization of the two common fragile sites present in bands 1p31.2 and 1p32, the expression of which is increased in patients with neuroblastomas.
Chromosome Mapping↗
Multipoint linkage analysis and homogeneity tests in 15 Dutch X-linked retinitis pigmentosa families.
Linkage analysis and homogeneity tests were carried out in 15 Dutch families segregating X-linked retinitis pigmentosa (X L R P). The study included segregation data for eight polymorphic DNA markers from the short arm of the human X chromosome. The results of both multipoint linkage analysis in individual families and heterogeneity analysis support the view that there are only two X L R P loci on the short arm of the human X chromosome, with one locus near the OTC gene and one in the vicinity of DXS255. Furthermore, our data confirm the hypothesis that a tapetal reflex in female carriers can be observed more frequently, if not exclusively, in X L R P families of the R P 3 type.
Adolescent↗
A polymorphic STS in intron 44 of the dystrophin gene.
A 300-bp EcoRV polymorphism, detected with P20 (DXS269) in intron 44 of the human dystrophin gene, is due to an insertion or deletion. To make this restriction fragment length polymorphism (RFLP) available for polymerase chain reaction (PCR) analysis, we sequenced both alleles of this polymorphism and synthesized primers flanking the mutation site. The origin of the mutation is a single Alu repeat insertion. The 300-bp polymorphism can now be successfully detected by PCR and provides an excellent tool to detect female carriers in this deletion prone region of the dystrophin gene.
Base Sequence↗
A CA-repeat polymorphism near DXS418 (P122).
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Adenine↗
Dynamic mutation in Dutch Huntington's disease patients: increased paternal repeat instability extending to within the normal size range.
Analysis of the distribution of normal and expanded alleles of the polymorphic (CAG)n repeat in the IT15 gene in the Dutch population confirmed the presence of an expanded repeat on all Huntington's disease (HD) chromosomes. Our results show that the size distributions of normal and affected alleles overlap. Normal alleles range from 11 to 37 repeats and HD alleles contain 37 to 84 repeats. A clear correlation is found between age at onset and repeat length, but the spread of the age at onset in the major repeat range producing characteristic HD is too wide to be of diagnostic value. In the available parent-offspring pairs, maternal HD alleles show a moderate instability with a slight preponderance of size increase over size decrease. Paternal alleles have a bimodal distribution: the majority (69%) behave similarly to the maternal alleles, while the remainder (31%) show a dramatic expansion, the degree of which appears proportional to the initial size. This is shown in three out of four juvenile patients, who have repeats of 71, 74, and 84 copies, respectively, originating from expanded paternal HD alleles in the previous generation. Two sporadic cases are caused by expansion of 'large' normal paternal alleles of 32 and 34 repeats, respectively, to 46 copies. This not only confirms the diagnosis of HD in two de novo cases, but it also underlines the increased paternal instability. In addition paternal repeat instability was once detected within the normal range in two sibs who inherited 21 and 22 repeats, respectively, on the same paternal chromosome. In two Dutch HD families the segregation of the expanded (CAG)n repeat was found. Analysis of the (CAG)n repeat in our previously reported recombinants confirmed their disease status.
Adult↗
Characterization and cell type distribution of a novel, major transcript of the Duchenne muscular dystrophy gene.
Previously we identified a novel 6.5 kb mRNA transcribed from the Duchenne muscular dystrophy (DMD) gene. This mRNA differs in coding content and tissue distribution from the known muscle type and brain type 14 kb DMD mRNAs which code for dystrophin. The novel transcript shares with dystrophin most of the sequence coding for the cysteine-rich and C-terminal domains. Here we used cDNA cloning to identify the divergence point between the common region and the sequence unique to the novel mRNA at the 5' end of the sequence encoding the cysteine-rich domain of dystrophin. This unique sequence containing the translation initiation site is located in a new exon in the intron between exons 62 and 63 of the dystrophin gene. Using probes containing RNA sequences specific to the novel mRNA, we investigated the expression of this mRNA in various tissues and cell types. The study reveals that this mRNA is the main DMD gene product detectable in a variety of nonmuscle tissues including brain cells. The amount of this mRNA in some tissues is comparable to the amount of dystrophin mRNA in the muscle. The expression of the 6.5 kb mRNA is down-regulated during differentiation of myogenic cells; it is present in small amounts in proliferating myoblasts but is undetected in differentiated muscle cultures depleted of mononucleated cells.
Animals↗
Isolation and characterization of cell hybrids containing human Xp-chromosome fragments.
We have subjected C12D, a Chinese hamster hybrid containing only the human X chromosome, to 6-thioguanine selection. The majority of the derivative clones retained rearranged Xp-fragments, which were characterized by using a combination of enzyme markers, DNA probes, and in situ hybridization. Two of these, TG2 and TG5sc9.1, contained only an Xpter----p21 fragment and should be an ideal resource for directed cloning from this region. A possible mechanism for the specific retention of Xp-fragments is discussed.
Animals↗