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PubMed1989

Molecular cloning of human telomeres in yeast.

Telomeres are the DNA sequences found at the ends of linear chromosomes. They define the boundaries of the genetical and physical maps of such chromosomes and so are particularly important for the complete mapping of large genomes that is now being attempted. Telomeres have been intensively studied in the yeast Saccharomyces cerevisiae and in ciliated protozoa: in these organisms the telomeric DNA consists of arrays of tandemly repeated short sequences in which one strand is guanosine-rich and oriented 5' to 3' towards the chromosome end. The conservation of these structural features is reflected in the observation that telomeric DNA from a variety of protozoa will function as telomeres on artificial linear mini-chromosomes in yeast. Tandem arrays of the sequence TTAGGG have been identified at the telomeres of humans and other mammals and also of trypanosomes. This indicates that the structural features of telomeres are conserved between higher and lower eukaryotes and implies that human telomeric DNA could function in yeast. I have used this idea to develop a strategy to isolate a specific human telomere as a molecular clone in yeast and have devised a simple and effective way of cloning other human telomeres and their associated sequences.

Animals↗
PubMed1998

Monitoring of lineage-specific chimaerism allows early prediction of response following donor lymphocyte infusions for relapsed chronic myeloid leukaemia.

Donor lymphocyte infusions (DLI) have been shown to enhance the graft-versus-leukaemia (GVL) effect and induce haematological and molecular remission in patients with relapsed CML following allogeneic bone marrow transplantation (BMT). The potent donor cell-mediated cytolysis following DLI may lead to a short period of aplasia before the re-establishment of donor haematopoiesis. The absence of detectable donor cells in patients prior to DLI infusion may result in permanent aplasia in certain patients. We report on four patients who relapsed 1, 3, 6.5 and 7 years post-BMT for chronic phase CML and were treated with DLI from their original BMT donor. Polymorphic short tandem repeats (STRs) were used to assess haematological chimaerism both prior to and following DLI. At the time of relapse, STR-PCR indicated the presence of donor cells in all four patients, at levels ranging from 1-40%. A clinical and molecular response was seen in 4/4 patients following a short period of cytopenia and all patients remain in clinical remission with a follow-up of 2 months-3 years post-DLI. STR-PCR indicated that a response was occurring during the period of pancytopenia when metaphase analysis was unsuccessful. Lineage-specific analysis of the cellular response to DLI was monitored using STR-PCR of peripheral blood (PB) and bone marrow (BM) lymphocyte-enriched fractions and CD2-positive and -negative T cell fractions. In one patient BM and PB CD34-positive and -negative fractions were also assessed. A change in the ratio of donor:recipient cells in the PB lymphocyte fraction was the earliest molecular indication of an anti-leukaemic response. Subsequent conversion to donor chimaerism occurred in the other lineages and the granulocyte fraction was the last lineage to convert. In conclusion, lineage-specific STR-PCR permits detailed monitoring of subtle changes in donor/recipient cell dynamics in specific lineages following DLI during the crucial pancytopenic phase and may be a useful predictor of haematological response to DLI therapy.

Adult↗
PubMed2005

Study of six X-linked tetranucleotide microsatellites: population data from five Spanish regions.

We studied six X-linked microsatellites in a large group of Spanish individuals (n=614) from five different regions located in northern, central and southern Spain. All the markers had tetranucleotide repeat units (DXS9895, DXS9898, DXS7130, DXS7132, GATA172D05 and DXS6789). They were amplified in two triplex PCR reactions. There were no significant sex- or region-related differences in allelic frequencies, suggesting that general national databases can be adequate as a reference for X-linked markers. The analysis of those six short tandem repeats combined in 316 males revealed 300 different "temporary haplotypes", 283 of which were found only once. There was no evidence for statistically significant linkage disequilibrium among the loci studied. Therefore these markers are quite polymorphic and useful for forensic purposes.

Chromosomes, Human, X↗
PubMed2003

Molecular characterization and Austrian Caucasian population data of the multi-copy Y-chromosomal STR DYS464.

DYS464 is a multi-copy STR system with four positions on the Y-chromosome (DYS464a, b, c, and d) which was recently identified and characterized [Forensic Sci. Int. 130 (2002) 97]. The aims of our study were to perform a population study, to estimate the mutation rate and an extensive sequence analysis in order to confirm the nomenclature. Fourteen different alleles were found in an Austrian population sample with an allele length varying from 9 to 19 repeats. All alleles were cloned and sequenced. Alleles 9-19 showed the general repeat structure (CCTT)n...(CCTT)2...(CCTT)3...(CCTT)4...(CCTT)2...(CCTT)2. The nomenclature is based on the number of repeated units of the variable (CCTT)n-stretch only. In 13% of the samples intermediate alleles, namely 14.3A, 14.3B and 15.3 were detected. In these alleles the variable repeat block is interrupted by a CTT motif (14.3A: (CCTT)3CTT(CCTT)11; 14.3B and 15.3: (CCTT)7CTT(CCTT)7/8). A comparison with GenBank entries revealed the existence of a length variant due to a deletion of one cytosine in the 5' flanking region of the first repeat block. We designed an alternative forward primer to circumvent possible ambiguities in the allele designation. A total of 54 different genotypes were identified in 135 men corresponding to a discrimination capacity (DC) of 40% and a gene diversity (GD) of 0.97. These values are much higher than those of other Y-chromosomal short tandem repeats (Y-STRs). DYS464 has the same haplotype diversity (HD) as the combination of the five Y-STR loci with the lowest gene diversities of the Y-STR core set. On the other hand, a combination of the three most diverse loci (DYS464, DYS385 and DYS390) has the same capacity to distinguish between paternal lineages than the complete minimal haplotype (minHT) consisting of eight Y-STR loci. In our population sample the addition of DYS464 to the minHT increases the number of different haplotypes from 110 to 122. The mutation-rate estimate based on the 70 meioses analyzed amounts to 2.86 x 10(-2) (95% confidence interval 3.5 x 10(-3) to 9.95 x 10(-2)). This value is approximately 10 times higher than the average mutation-rate estimate for Y-STRs.

Alleles↗
PubMed1998

Phylogenetic resolution of complex mutational features at Y-STR DYS390 in aboriginal Australians and Papuans.

Y-chromosomal short tandem repeats (STRs) are used for the study of male aspects of human evolution as well as for forensic applications and paternity testing. Both applications require an understanding of the underlying mutational mechanisms that create variability. We describe complex mutations at the substructured DYS390 STR locus in 97 natives of the New Guinea/Australian region. Sequencing of short alleles in these populations indicates multirepeat deletions. All samples are further characterized using the five additional Y-STR loci DYS19, DXYS156-Y, DYS391, DYS392, and DYS393. Phylogenetic analysis of the resulting haplotypes yields ethnically specific clusters predating the settlement of Australia and Papua New Guinea (although archaic Homo sapiens or Homo erectus lineages are absent). The phylogeny confirms that DYS390 violates the stepwise mutation model and demonstrates that the DYS390 locus mutates relatively rapidly and retains its variability after structural change.

Alleles↗
PubMed2003

Reduced-intensity rituximab-BEAM-CAMPATH allogeneic haematopoietic stem cell transplantation for follicular lymphoma is feasible and induces durable molecular remissions.

The indolent non-Hodgkin's lymphomas are theoretically curable through allogeneic haematopoietic stem cell transplantation (allo-HSCT). The applicability of standard conditioning allo-HSCT is, however, restricted by its toxicity. Recently, reduced-intensity conditioning regimens have demonstrated efficacy with significantly reduced early morbidity and mortality. We examined the safety and efficacy of rituximab-BEAM-CAMPATH as reduced-intensity conditioning for allo-HSCT in follicular lymphomas. Minimal residual disease was assessed by polymerase chain reaction (PCR) for bcl-2/IgH translocations, and chimerism by X,Y-FISH or PCR amplification of short tandem repeat sequences. At a median follow-up of 521 days (371-719), four of five patients were alive and three were in complete molecular remission. Three patients required pre-emptive treatment for CMV reactivation. One succumbed to a perforated viscus and one had slowly progressive disease. A graft-versus-lymphoma effect was demonstrable and quantitative PCR for bcl-2/IgH may allow better accuracy in scheduling post-allograft rituximab and/or donor lymphocyte infusions. Although follow-up is short, reduced-intensity allo-HSCT with BEAM-CAMPATH conditioning appears to be safe, effective in inducing a molecular remission and is potentially curative. Further recruitment and much longer follow-up will be necessary to determine if this impacts favourably upon disease-free and overall survival.

Adult↗
PubMed1995

In utero paternity testing following alleged sexual assault. A comparison of DNA-based methods.

OBJECTIVE: To compare DNA typing by both variable number of tandem repeats (VNTR) and polymerase chain reaction (PCR) methods to determine the utility of each for prenatal paternity testing following sexual assault. To consider ethical issues of limiting prenatal paternity studies. DESIGN: Criterion standard. SETTING: Prenatal diagnostic clinic after determination of pregnancy following an alleged sexual assault. SUBJECTS: Ten prenatal paternity cases accepted during a 5-year period. INTERVENTION: DNA-based paternity testing. MAIN OUTCOME MEASURE: Inclusion or exclusion of paternity by consensual partner. RESULTS: In all cases DNA typing using the PCR-based method provided the same conclusion as that from VNTR-based data. High probabilities of paternity were reported with both methods. CONCLUSIONS: DNA typing with PCR using short tandem repeat loci provides a reliable method for quickly determining paternity in prenatal cases. The ethics of providing paternity testing in the context of sexual assault is discussed. The issue of providing prenatal paternity testing in consensual relationships is considered.

Adolescent↗
PubMed2003

Myotonic dystrophy type 2: human founder haplotype and evolutionary conservation of the repeat tract.

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on either chromosome 19 (DM1) or 3 (DM2). In 2001, we demonstrated that DM2 is caused by a CCTG expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. To investigate the ancestral origins of the DM2 expansion, we compared haplotypes for 71 families with genetically confirmed DM2, using 19 short tandem repeat markers that we developed that flank the repeat tract. All of the families are white, with the majority of Northern European/German descent and a single family from Afghanistan. Several conserved haplotypes spanning >700 kb appear to converge into a single haplotype near the repeat tract. The common interval that is shared by all families with DM2 immediately flanks the repeat, extending up to 216 kb telomeric and 119 kb centromeric of the CCTG expansion. The DM2 repeat tract contains the complex repeat motif (TG)(n)(TCTG)(n)(CCTG)(n). The CCTG portion of the repeat tract is interrupted on normal alleles, but, as in other expansion disorders, these interruptions are lost on affected alleles. We examined haplotypes of 228 control chromosomes and identified a potential premutation allele with an uninterrupted (CCTG)(20) on a haplotype that was identical to the most common affected haplotype. Our data suggest that the predominant Northern European ancestry of families with DM2 resulted from a common founder and that the loss of interruptions within the CCTG portion of the repeat tract may predispose alleles to further expansion. To gain insight into possible function of the repeat tract, we looked for evolutionary conservation. The complex repeat motif and flanking sequences within intron 1 are conserved among human, chimpanzee, gorilla, mouse, and rat, suggesting a conserved biological function.

Adult↗
PubMed1994

Enzymatic elongation of microsatellite oligomers for use in direct-label chemiluminescent hybridizations.

Short, synthetic oligonucleotide sequences representing microsatellites and other short tandem repeats can be elongated (concatamerized) using a simple method in which complementary strands are annealed, phosphorylated, primer extended and ligated. When used in direct-label chemiluminescent hybridizations, the elongated microsatellite sequences provide an approximately 30-fold increase in signal strength compared with microsatellite oligomers that have not been concatamerized. Concatamerization of simple repeat oligomers further enables the use of relatively short oligonucleotide sequences in direct-label chemiluminescent hybridization experiments, thereby reducing the overall need for radioisotopes in certain commonly performed laboratory procedures such as DNA fingerprinting and selection of clones containing microsatellite sequences.

Animals↗
PubMed2005

Three cases with de novo 6q imbalance and variable prenatal phenotype.

We describe two families in which three fetuses had a de novo 6q imbalance and abnormal phenotypes. We determined the boundaries and the parental origin of the chromosomal alterations by segregation analysis using a panel of short tandem repeats (STRs) located on 6q. Cases 1 and 2 (family A) were two sibs with 6q imbalance involving different regions. Case 1 was a female fetus with arthrogryposis, who had a complex rearrangement resulting in two deleted regions (6q22 and 6q25.1-q25.2) and a duplication of 6q23-q25.1. This latter imbalance was reported previously and is associated with joint contractures and short neck, also present in this fetus. The sib (case 2) had intrauterine growth restriction (IUGR) and agenesis of the ductus venosus. This male died shortly after birth; postnatal karyotype and molecular investigations showed a 6q21 de novo deletion. Case 3 (family B) had a prenatally detected deletion of 6q14-q16. Autopsy of the fetus documented minor facial anomalies and contractures of the limbs. All rearrangements were de novo and of paternal origin. Our data and the consistent number of cases of de novo 6q alterations previously reported suggest that chromosome arm 6q could be prone to rearrangements resulting in heterogeneous phenotypes. In family A, chromosome 6q imbalances involving different chromosomal regions were present in two consecutive pregnancies. In such cases counseling should suggest the impossibility of excluding recurrence of a chromosomal imbalance, and should discuss the option of early prenatal diagnosis in subsequent pregnancies.

Chromosome Aberrations↗
PubMed2002

Application of DNA fingerprinting in medicolegal practice.

Fingerprinting is thought to establish the identify of an individual in forensic cases. The technique is extensively used for forensic purposes. Deoxyribonucleic acid (DNA) is the vehicle of generational transference of heritable unit. While arching markers for genetic disease professor Alec Jeffreys discovered that certain regions of DNA showed variations in the number of tandem repeats known as variable number of tandem repeats (VNTRs). Thus DNA fingerprint was named by observing the number of repeated sequences which differ from individual to individual. The structure of DNA is quite flexible, within the nucleus of each cell resides an identical copy of the individual's genetic material, DNA. The coding regions of the genomic DNA are known as genes. The DNA fingerprinting in forensic science has generated considerable excitement in the criminal justice community. DNA fingerprinting can be applied to identify an individual in criminal and civil cases. Polymerase chain reaction has revolutionised molecular biology it has an ability to amplify (usually fewer than 3000 bp) a particular sequence of DNA into million of copies in a very short period. Consequently only a very tiny amount of an organism's DNA needs to be available originally. This property of polymerase chain reaction has enabled to analyse many forensic samples particularly which are degraded. Microsatellite DNA or commonly as short tandem repeats are scattered throughout the human genome and occur on an average of every 10,000 nucleotides. Microsatellite markers are considered to be the most powerful genetic markers. Collection, preservation and handling are the integral part of DNA fingerprinting analysis. There are various methods to isolate DNA from different biological materials but presently most of the laboratories prefer using FTA paper. The age of humans can be estimated by using DNA based on telomere shortening.

Clinical Medicine↗
PubMed1997

Fluorescence imaging in human identity testing.

We have investigated the use of fluorescence detection and the FluorImager S1 System (Molecular Dynamics) for analyzing a comprehensive set of human DNA typing tests. We used an alkaline phosphatase-conjugated YNH24 oligonucleotide probe to the repeat-containing D2S44 locus to detect both alleles in 50 ng of human genomic DNA (0.025 amol) by Southern hybridization using a chemifluorescent substrate. We used a similar approach to quantify human DNA using an enzyme-conjugated oligonucleotide probe to the D17Z1 locus. Both fluorescent nucleic acid gel staining and direct fluorescent labeling methods were tested to detect PCR-based D1S80 and short tandem repeat (STR) multiplex allele profiles. The fluorescent staining method sensitively detected these allelic profiles in both denaturing and non-denaturing acrylamide gels using a simple, 10-min procedure. Fluorescent primers eliminate the doublet band patterns often seen with staining methods, which label both strands of the amplified products. This complicates interpretation of STR typing tests. Only one primer for each locus is labeled, so only one strand of the DNA product is detected. Fluorescein end-labeled primers were used in multiplex PCR to amplify, detect and type STRs.

Benzothiazoles↗
PubMed1997

Trinucleotide repeat polymorphism within exon 5 of the MICA gene (MHC class I chain-related gene A): allele frequency data in the nine population groups Japanese, Northern Han, Hui, Uygur, Kazakhstan, Iranian, Saudi Arabian, Greek and Italian.

We recently identified a trinucleotide repeat polymorphism, (GCT)n, within the transmembrane (TM) segment of the human MHC class I MICA gene (MHC class I chain-related gene A). Five distinct alleles (A4, A5, A5.1, A6, A9) corresponding to 4, 5, 5 with one nucleotide insertion, 6 and 9 repetitions, respectively, have been detected in various HLA-homozygous B cell lines. Here we present allele frequencies for this trimeric short tandem repeat (STR) in 604 unrelated individuals collected from nine human populations (Japanese, Northern Han, Hui, Uygur, Kazakhstan, Iranian, Saudi Arabian, Greek and Italian) determined using the polymerase chain reaction (PCR) combined with fluorescent-based automated fragment detection technology. All alleles were present in each population, but allelic distributions varied from one population to another. No new alleles (such as A7 or A8) were identified. The evolutionary and structural significance of these data as well as the potential application to forensic medicine is discussed.

Alleles↗
PubMed2003

Allogeneic peripheral blood stem cell transplantation in the treatment of severe aplastic anemia and severe infection.

OBJECTIVE: To investigate the efficacy of allogeneic peripheral blood stem cell transplantation (PBSCT) in the treatment of severe aplastic anemia (SAA) and severe infection. METHODS: A patient with SAA and pseudomonas aeruginosa septicemia was treated with PBSCT from an HLA-identical sibling with cyclophosphamide (CY) and total body irradiation (TBI) for conditioning. The patient was infused with 20.3 x 10(8)/kg mononuclear cells including 61.0 x 10(6)/kg CD34(+) cells following the conditioning regimen. RESULTS: Twelve days after PBSCT, the absolute neutrophil count (ANC) of 1.0 x 10(9)/L was achieved, with platelet count > 50 x 10(9)/L at twenty days. The donor origin of engraftment was confirmed by polymerase chain reaction (PCR) analysis of short tandem repeats at the end of the first, sixth and twelfth month. The patient's body temperature dropped to normal level when her ANC reached 0.5 x 10(9)/L on day 10, and the bacterial culture of blood sample became negative subsequently. Symptoms and signs of acute or chronic graft versus host disease (GVHD) were not observed in 30 months after PBSCT. CONCLUSIONS: Hematopoiesis was reconstituted shortly after PBSCT. The combination of CY and TBI and the infusion of sufficient peripheral blood stem cells may contribute to the successful engraftment. PBSCT may be considered as the first choice when hematopoietic stem cell transplantation is needed for SAA patients complicated with severe infection.

Adult↗
PubMed2001

Successful application of nonmyeloablative transplantation for paroxysmal nocturnal hemoglobinuria.

OBJECTIVE: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder that manifests as hemolytic anemia, venous thrombosis, and deficient hematopoiesis. Although allogeneic hematopoietic stem cell transplantation is considered the only curative therapeutic measure, transplant-related mortality is not negligible. Several studies supported the use of nonmyeloablative stem cell transplantation (NST) for patients of advanced age or with organ dysfunction. Hence, we used NST in a PNH patient who suffered from acute renal failure due to repeated episodes of hemolysis. MATERIALS AND METHODS: We performed NST using a conditioning regimen consisting of cladribine 0.11 mg/kg x 6, busulfan 4 mg/kg x 2, and rabbit anti-thymocyte globulin 2.5 mg/kg x 2. He received peripheral blood stem cells from his human leukocyte antigen-matched brother. Prophylaxis against graft-vs-host disease was performed with cyclosporine A alone. Chimerism of peripheral blood mononuclear cells was evaluated serially using short tandem repeat analysis and flow cytometry. RESULTS: No meaningful regimen-related toxicities were documented. Donor chimerism of 90 to 100% was achieved on day 14 and thereafter. The patient is doing well, without any recurrence of hemolysis 6 months after transplant. Follow-up chimerism studies confirmed stable and functioning donor-type hematopoiesis. CONCLUSIONS: NST may become a safe and curative approach in patients with PNH. Further studies are needed to establish the role of NST for treatment of PNH.

Antilymphocyte Serum↗
PubMed2000

Long microsatellite alleles in Drosophila melanogaster have a downward mutation bias and short persistence times, which cause their genome-wide underrepresentation.

Microsatellites are short tandemly repeated DNA sequence motifs that are highly variable in most organisms. In contrast to mammals, long microsatellites (>15 repeats) are extremely rare in the Drosophila melanogaster genome. To investigate this paucity of long microsatellites in Drosophila, we studied 19 loci with exceptionally long microsatellite alleles. Inter- and intraspecific analysis showed that long microsatellite alleles arose in D. melanogaster only very recently. This lack of old alleles with many repeats indicated that long microsatellite alleles have short persistence times. The size distribution of microsatellite mutations in mutation-accumulation lines suggests that long alleles have a mutation bias toward a reduction in the number of repeat units. This bias causes the short persistence times of long microsatellite alleles. We propose that species-specific, size-dependent mutation spectra of microsatellite alleles may provide a general mechanism to account for the observed differences in microsatellite length between species.

Alleles↗
PubMed2003

The tetranucleotide repeat polymorphism C2_4_4: population data and linkage disequilibria with HLA class I.

The tetranucleotide repeat locus C2_4_4 situated in the HLA class I region (6p21.3) and the HLA-ABC specificities were investigated in an Austrian population sample of 240 unrelated Caucasoid individuals. The analysis of the linkage disequilibrium between C2_4_4 and HLA class I showed several significant values, especially when factors coded for by so-called "superhaplotypes" were considered; such linkage disequilibria are of importance for the practical use of HLA coded short tandem repeats.

Austria↗
PubMed1995

Somatic mutations in VNTR-locus D1S7 in human colorectal carcinomas are associated with microsatellite instability.

To elucidate mutation mechanisms in hypervariable VNTR loci, we have studied somatic mutation events with the minisatellite probe MS1 (VNTR locus D1S7) in 224 colorectal carcinomas (CRC). The D1S7 locus consists of a 9-basepair (bp) repeat unit. The copy number varies from about 100 to 2000, and the germline mutation rate is high. Here we demonstrate a high D1S7 somatic mutation rate in CRC (37/224), higher than indicated earlier by others. We also demonstrate that the most frequent mutational event by far (n = 34) involves small reductions in VNTR fragment size (median loss 22 repeat units, range 2-154), furthermore, in one-half of these cases, this event is biallelic. We wanted to test whether these somatic mutations mirror the same genetic instability as seen by RER (replication error), a phenomenon recently described in tumour DNA from both sporadic and familial cases of CRC. All blood/tumour DNA pairs displaying MS1 mutation (n = 37) as well as 37 randomly selected pairs without MS1 mutation were tested with four tetranucleotide short tandem repeats (STRs, microsatellites). There is a strong association between mutations at the D1S7 locus and the occurrence of new STR alleles (P < 0.001). This is the first report of the existence of a minisatellite as a marker for genetic instability/RER in colorectal carcinomas. The findings may also cast light upon the mechanism for somatic mutations in this minisatellite.

Adenocarcinoma↗