John A.L. Armour

Uniparental paternal disomy in Angelman's syndrome

Angelmans syndrome and Prader-Willi syndrome are both causes of mental retardation with recognisable, but quite different, clinical phenotypes. Both are associated with deletions of chromosome 15q11-13, of maternal origin in Angelmans and paternal in Prader-Willi. Prader-Willi can arise by inheritance of two chromosomes 15 from the mother and none from the father (uniparental maternal disomy). In 2 patients with Angelmans syndrome we found evidence of uniparental paternal disomy. The phenotypic effects of maternal and paternal disomy of chromosome 15 are very different and inheritance of two normal 15s from one parent does not lead to normal development--strong evidence in man for genomic imprinting, in which the same gene has different effects dependent upon its parental origin.

The frequency of uniparental disomy in Prader-Willi syndrome: Implications for molecular diagnosis

BACKGROUNDnPrader-Willi syndrome is a genetic disorder characterized by infantile hypotonia, obesity, hypogonadism, and mental retardation, but it is difficult to diagnose clinically in infants and young children. In about two thirds of patients, a cytogenetically visible deletion can be detected in the paternally derived chromosome 15 (15q11q13). Recently, patients with Prader-Willi syndrome have been described who do not have the cytogenetic deletion but instead have two copies of the 15q11q13 region that are inherited from the mother (with none inherited from the father). This unusual form of inheritance is known as maternal uniparental disomy. Using molecular genetic techniques, we sought to determine the frequency of uniparental disomy in Prader-Willi syndrome.nnnMETHODSnWe performed molecular analyses using DNA markers within 15q11q13 and elsewhere on chromosome 15 in 30 patients with Prader-Willi syndrome who had no cytogenetically visible deletion. We also studied their parents. Three patients with Prader-Willi syndrome who had a cytogenetic deletion served as controls.nnnRESULTSnIn 18 of the 30 patients without a cytogenetic deletion (60 percent), we demonstrated the presence of maternal uniparental disomy for chromosome 15 and its association with advanced maternal age. In another eight patients (27 percent), we identified large molecular deletions. The remaining four patients (13 percent) had evidence of normal biparental inheritance for chromosome 15; three of these patients were the only ones in the study who had some atypical clinical features.nnnCONCLUSIONSnIn about 20 percent of all cases, Prader-Willi syndrome results from the inheritance of both copies of chromosome 15 from the mother (maternal uniparental disomy). With the combined use of cytogenetic and molecular techniques, the genetic basis of Prader-Willi syndrome can be identified in up to 95 percent of patients.

SYSTEMATIC CLONING OF HUMAN MINISATELLITES FROM ORDERED ARRAY CHAROMID LIBRARIES

We present a rapid and efficient method for the isolation of minisatellite loci from human DNA. The method combines cloning a size-selected fraction of human MboI DNA fragments in a charomid vector with hybridization screening of the library in ordered array. Size-selection of large MboI fragments enriches for the longer, more variable minisatellites and reduces the size of the library required. The library was screened with a series of multi-locus probes known to detect a large number of hypervariable loci in human DNA. The gridded library allowed both the rapid processing of positive clones and the comparative evaluation of the different multi-locus probes used, in terms of both the relative success in detecting hypervariable loci and the degree of overlap between the sets of loci detected. We report 23 new human minisatellite loci isolated by this method, which map to 14 autosomes and the sex chromosomes.

Analysis of somatic mutations at human minisatellite loci in tumors and cell lines.

Hypervariable human minisatellite loci show a substantial level of germline instability, and spontaneous mutation rates to new length alleles have been measured directly by pedigree analysis. We now show that mutation events altering the number of minisatellite repeat units are not restricted to the germline, but also arise in other tissues. Mutant alleles can be detected at a very low frequency in lymphoblastoid cell lines and at much higher frequencies in clonal tumor cell populations, most particularly in gastrointestinal adenocarcinomas. Mutant alleles in these tumors are usually present at a dosage equal to or greater than that of the progenitor allele, indicating that most or all of the tumor cells carry the same clonally derived mutant allele. As with germline mutation, the incidence of somatic mutations in tumors varies from locus to locus, with the same locus showing the highest level of germline and somatic instability. Most length changes, as those in the germline, are of only a few repeat units; however, very large changes are also observed, implying that such mutations can occur in the absence of meiosis.

REPORT on the Third International Workshop on Chromosome 9

summary The Fifth International workshop on chromosome 9 comprised a gathering of 36 scientists from seven countries and included a fairly even distribution of interests along chromosome 9 as well as a strong input from more global activities and from comparative mapping. At least eight groups had participated in the goal set at the previous workshop which was to improve the fine genetic mapping in dierent regions of chromosome 9 by meiotic breakpoint mapping in allocated regions and this has resulted in some greatly improved order information. Excellent computing facilities were available and all contributed maps were entered not only into SIGMA (and thence submitted to GDB) but also into a dedicated version of ACEDB which can be accessed on the Web in the form of one of 28 slices into which the chromosome has been arbitrarily divided. It was generally agreed that the amount of data is now overwhelming and that the integration and validation of all data is not only unrealistic in a short meeting but probably impossible until the whole chromosome has been sequenced and fully annotated. Sequence-ready contigs presented at the meeting totalled about 3 MB which is about one fiftieth of the estimated length. The single biggest barrier to integration of maps is the problem of non-standard nomenclature of loci. In the past 2 workshops eorts have been made to compare traditional ‘consensus’ maps made by human insight (still probably best for small specific regions) with those generated with some computer assistance (such as SIGMA) and those generated objectively by defined computer algorithms such as ldb. Since no single form of map or representation is entirely satisfactory for all purposes the maps reproduced in the published version of the report are confined to one of the genetic maps, in which Genethon and older markers have been incorporated, a Sigma map of the genes as symbols together with a listing of known ‘disease’ genes

Biology and applications of human minisatellite loci.

Highly repetitive minisatellites include the most variable human loci described to date. They have proved invaluable in a wide variety of genetic analyses, and despite some controversies surrounding their practical implementation, have been extensively adopted in civil and forensic casework. Molecular analysis of internal allelic structure has provided detailed insights into the repeat-unit turnover mechanisms operating in germline mutations, which are ultimately responsible for the extreme variability seen at these loci.

Hypervariable minisatellite DNA sequences in the Indian peafowl Pavo cristatus.

We report here for the first time the large-scale isolation of hypervariable minisatellite DNA sequences from a non-human species, the Indian peafowl (Pavo cristatus). A size-selected genomic DNA fraction, rich in hypervariable minisatellites, was cloned into Charomid 9-36. This library was screened using two multilocus hypervariable probes, 33.6 and 33.15 and also, in a probe-walking approach, with five of the peafowl minisatellites initially isolated. Forty-eight positively hybridizing clones were characterized and found to originate from 30 different loci, 18 of which were polymorphic. Five of these variable minisatellite loci were studied further. They all showed Mendelian inheritance. The heterozygosities of these loci were relatively low (range 22-78%) in comparison with those of previously cloned human loci, as expected in view of inbreeding in our semicaptive study population. No new length allele mutations were observed in families and the mean mutation rate per locus is low (less than 0.004, 95% confidence maximum). These loci were also investigated by cross-species hybridization in related taxa. The ability of the probes to detect hypervariable sequences in other species within the same avian family was found to vary, from those probes that are species-specific to those that are apparently general to the family. We also illustrate the potential usefulness of these probes for paternity analysis in a study of sexual selection, and discuss the general application of specific hypervariable probes in behavioral and evolutionary studies.

Report and abstracts of the Second International Workshop on Human Chromosome 9 Mapping 1993.

The Second International Workshop on Human Chromosome 9 was held in Chatham, Massachusetts on April 18--20, 1993. Fifty-three abstracts were received and the data presented on posters. The purpose of the meeting was to bring together all interested investigators working on the map of chromosome 9, many of whom had disease-specific interests. After a brief presentation of interests and highlighted results, the meeting broke up into the following subgroups for production of consensus maps: 9p; 9cen-q32; 9q32 ter. A global mapping group also met. Reports of each of these working groups is presented in the summary.

Principles and Recent Advances in Human DNA Fingerprinting

Since 1985, DNA typing systems have played an increasingly important role in many aspects of human genetics, most notably in forensic and legal medicine. This article reviews the development of multilocus and single locus minisatellite DNA probes, and more recently the use of PCR to amplify hypervariable DNA loci, as well as discussing the biological properties of the unstable regions of DNA which form the basis of almost all DNA fingerprinting systems.

Human minisatellite alleles detectable only after PCR amplification

We present evidence that a proportion of alleles at two human minisatellite loci is undetected by standard Southern blot hybridization. In each case the missing allele(s) can be identified after PCR amplification and correspond to tandem arrays too short to detect by hybridization. At one locus, there is only one undetected allele (population frequency 0.3), which contains just three repeat units. At the second locus, there are at least five undetected alleles (total population frequency 0.9) containing 60-120 repeats; they are not detected because these tandem repeats give very poor signals when used as a probe in standard Southern blot hybridization, and also cross-hybridize with other sequences in the genome. Under these circumstances only signals from the longest tandemly repeated alleles are detectable above the nonspecific background. The structures of these loci have been compared in human and primate DNA, and at one locus the short human allele containing three repeat units is shown to be an intermediate state in the expansion of a monomeric precursor allele in primates to high copy number in the longer human arrays. We discuss the implications of such loci for studies of human populations, minisatellite isolation by cloning, and the evolution of highly variable tandem arrays.