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J S Wainscoat

Publications and source records attributed to J S Wainscoat.

At least 19 recordsLinked to original sources
PubMed2001

A novel gene, NSD1, is fused to NUP98 in the t(5;11)(q35;p15.5) in de novo childhood acute myeloid leukemia.

The recurrent translocation t(5;11)(q35;p15.5) associated with a 5q deletion, del(5q), has been reported in childhood acute myeloid leukemia (AML). We report the cloning of the translocation breakpoints in de novo childhood AML harboring a cryptic t(5;11)(q35;p15.5). Fluorescence in situ hybridization (FISH) analysis demonstrated that the nucleoporin gene (NUP98) at 11p15.5 was disrupted by this translocation. By using 3'--rapid amplification of complementary DNA ends (3'-RACE) polymerase chain reaction, we identified a chimeric messenger RNA that results in the in-frame fusion of NUP98 to a novel gene, NSD1. The NSD1 gene has 2596 amino acid residues and a 85% homology to the murine Nsd1 with the domain structure being conserved. The NSD1 gene was localized to 5q35 by FISH and is widely expressed. The reciprocal transcript, NSD1-NUP98, was also detected by reverse transcriptase--polymerase chain reaction. This is the first report in which the novel gene NSD1 has been implicated in human malignancy. (Blood. 2001;98:1264-1267)

Acute Disease↗
PubMed2001

Technical optimization of RhD zygosity determination by real-time quantitative polymerase chain reaction: implication for fetal RhD status determination by maternal plasma.

We have recently reported the development of a multiplex, real-time quantitative polymerase chain reaction (PCR) assay for RhD zygosity determination based on the coamplification and quantification of RHD-specific sequences in relation to a reference gene, albumin. This paper discusses the optimization procedure and technical parameters of this multiplex assay.

Albumins↗
PubMed2001

Detection of mammaglobin mRNA in the plasma of breast cancer patients.

It has been suggested that mammaglobin may be a useful marker for breast cancer clinical research. Studies investigating the detection of mRNA by RT-PCR from circulating carcinoma cells in the peripheral blood of breast cancer patients have shown that mammaglobin is a highly specific marker and correlates with several prognostic factors, such as lymph node involvement. We aimed to detect cell-free mammaglobin mRNA in the plasma of breast cancer patients and to investigate whether it can be used as a marker for diagnosis of breast cancer.

Biomarkers, Tumor↗
PubMed2001

Detection of Plasmodium falciparum DNA in plasma.

We present a new application in the field of circulating nucleic acids in plasma: the detection of parasite DNA in plasma. We have investigated the presence of Plasmodium falciparum (P. falciparum) DNA in the blood cells and plasma of patients with malaria. Ten blood samples from malaria patients and 9 blood samples from healthy volunteers were collected. Plasma was separated and DNA was extracted from both plasma and blood cells. DNA samples were subjected to a nested polymerase chain reaction (PCR) for the small subunit rRNA gene of P. falciparum and a control amplification of the human rRNA gene. All 6 cases positive by microscopy for P. falciparum were positive by PCR on DNA extracted from blood cells and plasma. Two cases negative by microscopy for malaria were positive by PCR--the blood samples were taken from one of the cases at 7 days after successful treatment of malaria and the other case at the onset of the malaria illness. Two other cases were negative by microscopy and by PCR on blood cells and plasma, both after successful treatment of malaria. The quantitation of Plasmodium DNA in plasma may prove to be a useful prognostic measurement in malaria, and the detection of P. falciparum DNA in archival stored plasma samples may allow the reconstruction of the recent historic evolution of the parasite genome.

Animals↗
PubMed2001

Determination of RhD zygosity: comparison of a double amplification refractory mutation system approach and a multiplex real-time quantitative PCR approach.

BACKGROUND: Rh isoimmunization and hemolytic disease of the newborn still occur despite the availability of Rh immunoglobulin. For the prenatal investigation of sensitized RhD-negative pregnant women, determination of the zygosity of the RhD-positive father has important implications. The currently available molecular methods for RhD zygosity assessment, in general, are technically demanding and labor-intensive. Therefore, at present, rhesus genotype assessment is most commonly inferred from results of serological tests. The recent elucidation of the genetic structure of the prevalent RHD deletion in Caucasians, as well as the development of real-time PCR, allowed us to explore two new approaches for the molecular determination of RhD zygosity. METHODS: Two methods for RhD zygosity determination were developed. The first was based on the double Amplification Refractory Mutation System (double ARMS). The second was based on multiplex real-time quantitative PCR. For the double ARMS assay, allele-specific primers were designed to directly amplify the most prevalent RHD deletion found in RhD-negative individuals in the Caucasian population. The multiplex real-time quantitative PCR assay, on the other hand, involved coamplification and quantification of RHD-specific sequences in relation to a reference gene, albumin, in a single PCR reaction. A ratio, DeltaCt, based on the threshold cycle, was then determined and reflects the RHD gene dosage. RESULTS: The allele-specific primers of the double ARMS assay reliably amplified the RHD-deleted allele and therefore accurately distinguished homozygous from heterozygous RhD-positive samples. The results were in complete concordance with serological testing. For the multiplex real-time quantitative PCR assay, the DeltaCt values clearly segregated into two distinct populations according to the RHD gene dosage, with mean values of 1.70 (SD, 0.17) and 2.62 (SD, 0.29) for the homozygous and heterozygous samples, respectively (P: <0.001, t-test). The results were in complete concordance with the results of serological testing as well as with the double ARMS assay. CONCLUSION: Double ARMS and real-time quantitative PCR are alternative robust assays for the determination of RhD zygosity.

Albumins↗
PubMed2000

Telomere length shortening in chronic myelogenous leukemia is associated with reduced time to accelerated phase.

Telomere shortening is associated with disease evolution in chronic myelogenous leukemia (CML). We have examined the relationship between diagnostic telomere length and outcome in 59 patients with CML who entered into the MRC CMLIII Trial by Southern blot hybridization using the (TTAGGG)(4) probe. Age-adjusted telomere repeat array (TRA) reduction was found to significantly correlate with time from diagnosis to acceleration, such that patients with a larger TRA reduction entered the accelerated phase more rapidly (r = -0.50; P =.008). Cox-regression analysis for this group was suggestive of a relationship between a greater TRA-reduction and a shorter time to acceleration (P =.054). Age-adjusted TRA reduction did not significantly affect either the time to blast crisis or overall survival. Our results show that telomere shortening observed at the time of diagnosis in CML significantly influences the time to progress to the accelerated phase. The measurement of diagnostic TRA may prove to be clinically important in the selection of patients at high risk of disease transformation in CML.

Age Factors↗
PubMed2000

Transcription mapping of the 5q- syndrome critical region: cloning of two novel genes and sequencing, expression, and mapping of a further six novel cDNAs.

The 5q- syndrome is a myelodysplastic syndrome with the 5q deletion ¿del(5q) as the sole karyotypic abnormality. We are using the expressed sequence tag (EST) resource as our primary approach to identifying novel candidate genes for the 5q- syndrome. Seventeen ESTs were identified from the Human Gene Map at the National Center for Biotechnology Information that had no significant homology to any known genes and were assigned between DNA markers D5S413 and D5S487, flanking the critical region of the 5q- syndrome at 5q31-q32. Eleven of the 17 cDNAs from which the ESTs were derived (65%) were shown to map to the critical region of the 5q- syndrome by gene dosage analysis and were then sublocalized by PCR screening to a YAC contig encompassing the critical region. Eight of the 11 cDNA clones, upon full sequencing, had no significant homology to any known genes. Each of the 8 cDNA clones was shown to be expressed in human bone marrow. The complete coding sequence was obtained for 2 of the novel genes, termed C5orf3 and C5orf4. The 2.6-kb transcript of C5orf3 encodes a putative 505-amino-acid protein and contains an ATP/GTP-binding site motif A (P loop), suggesting that this novel gene encodes an ATP- or a GTP-binding protein. The novel gene C5orf4 has a transcript of 3.1 kb, encoding a putative 144-amino-acid protein. We describe the cloning of 2 novel human genes and the sequencing, expression patterns, and mapping to the critical region of the 5q- syndrome of a further 6 novel cDNA clones. Genomic localization and expression patterns would suggest that the 8 novel cDNAs described in this report represent potential candidate genes for the 5q- syndrome.

Amino Acid Sequence↗
PubMed2000

Combined immunophenotyping and FISH identifies the involvement of B-cells in 5q- syndrome.

The 5q- syndrome is a distinct subtype of myelodysplastic syndrome (MDS) characterized by refractory anemia, deletion of the long arm of chromosome 5, del(5q), as the sole cytogenetic abnormality, and a low frequency of transformation to acute leukemia. Using combined immunophenotyping and fluorescence in situ hybridization (FISH), studies were carried out on bone marrow smears of three 5q- syndrome cases to identify the cell lineages carrying the 5q deletion. In all three cases, the granulocytic, monocytic, and erythroid lineages possessed the del(5q) clonal marker, whereas the T-lymphocytes did not. Interestingly, in one case, cells of B-lymphoid lineage also showed the presence of the del(5q). This is the first report to date showing involvement of an acquired 5q deletion associated with MDS in B-cells. This result suggests that in some cases, MDS arises in a multipotent cell with a capacity to differentiate into both myeloid and lymphoid cells.

Adult↗
PubMed2000

Physical mapping of the human ATX1 homologue (HAH1) to the critical region of the 5q- syndrome within 5q32, and immediately adjacent to the SPARC gene.

The 5q- syndrome is a myelodysplastic syndrome with the 5q deletion as the sole karyotypic abnormality. The human ATX1 homologue (HAH1), encodes a copper-binding protein with a role in antioxidant defence. We have mapped this gene to the 3 Mb critical region of gene loss of the 5q- syndrome within 5q32, flanked by the genes for ADRB2 and IL12B, using gene dosage analysis. Fine physical mapping of the HAH1 gene within this genomic interval was then performed by screening YAC and BAC contigs spanning the critical region of the 5q- syndrome using PCR amplification. The HAH1 gene maps immediately adjacent to the SPARC gene at 5q32, and is flanked by the genetic markers D5S1838 and D5S1419. The HAH1 gene is expressed in haematological tissues and plays a role in antioxidant defence. Antioxidant levels are low in most cancers and the importance of antioxidant enzymes in cancer genesis is well recognised. Genomic localisation, function and expression would suggest that the HAH1 gene represents a candidate gene for the 5q-syndrome.

Carrier Proteins↗
PubMed1999

A new recurrent translocation, t(5;11)(q35;p15.5), associated with del(5q) in childhood acute myeloid leukemia. The UK Cancer Cytogenetics Group (UKCCG)

Partial deletion of the long arm of chromosome 5, del(5q), is the cytogenetic hallmark of the 5q-syndrome, a distinct subtype of myelodysplastic syndrome-refractory anemia (MDS-RA). Deletions of 5q also occur in the full spectrum of other de novo and therapy-related MDS and acute myeloid leukemia (AML) types, most often in association with other chromosome abnormalities. However, the loss of genetic material from 5q is believed to be of primary importance in the pathogenesis of all del(5q) disorders. In the present study, we performed fluorescence in situ hybridization (FISH) studies using a chromosome 5-specific whole chromosome painting probe and a 5q subtelomeric probe to determine the incidence of cryptic translocations. We studied archival fixed chromosome suspensions from 36 patients with myeloid disorders (predominantly MDS and AML) and del(5q) as the sole abnormality. In 3 AML patients studied, this identified a translocation of 5q subtelomeric sequences from the del(5q) to the short arm of an apparently normal chromosome 11. FISH with chromosome 11-specific subtelomeric probes confirmed the presence of 11p on the shortened 5q. Further FISH mapping confirmed that the 5q and 11p translocation breakpoints were the same in all 3 cases, between the nucleophosmin (NPM1) and fms-related tyrosine kinase 4 (FLT4) genes on 5q35 and the Harvey ras-1-related gene complex (HRC) and the radixin pseudogene (RDPX1) on 11p15.5. Importantly, all 3 patients with the cryptic t(5;11) were children: a total of 3 of 4 AML children studied. Two were classified as AML-M2 and the third was classified as M4. All 3 responded poorly to treatment and had short survival times, ranging from 10 to 18 months. Although del(5q) is rare in childhood AML, this study indicates that, within this subgroup, the incidence of cryptic t(5;11) may be high. It is significant that none of the 24 MDS patients studied, including 11 confirmed as having 5q-syndrome, had the translocation. Therefore, this appears to be a new nonrandom chromosomal translocation, specifically associated with childhood AML with a differentiated blast cell phenotype and the presence of a del(5q).

Adult↗
PubMed1999

Telomere length shortening is associated with disease evolution in chronic myelogenous leukemia.

We studied telomere length in the peripheral blood leukocyte samples of a large group of patients with chronic myelogenous leukemia (CML) by Southern blot hybridization using the (TTAGGG)4 probe. The average telomere length expressed as the peak telomere repeat array (TRA) of the peripheral blood samples obtained from a group of 34 healthy age-matched controls ranged between 7.6 and 10.0 kb and the mean peak TRA was 8.7 kb. Forty-one patients in the chronic phase of CML were studied; 32/41 (78%) showed telomere reduction (<7.6 kb) relative to age-matched controls and the mean peak TRA was 6.4 kb (range 4.0-10.6 kb). Serial samples were analysed from 12 patients at both chronic phase and during disease progression. The leukocyte DNA of all 12 patients in accelerated phase and/or blast crisis showed telomere reduction relative to age-matched controls and the mean peak TRA was 4.1 kb (range 3.0-5.4 kb). The peak TRA in the accelerated or blast phase was reduced compared with the corresponding paired sample in the chronic phase in all cases studied. These data show that a marked reduction in telomere length is associated with disease progression in CML.

Adult↗
PubMed1998

Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma.

BACKGROUND: The ability to determine fetal RhD Status noninvasively is useful in the treatment of RhD-sensitized pregnant women whose partners are heterozygous for the RhD gene. The recent demonstration of fetal DNA in maternal plasma raises the possibility that fetal RhD genotyping may be possible with the use of maternal plasma. METHODS: We studied 57 RhD-negative pregnant women and their singleton fetuses. DNA extracted from maternal plasma was analyzed for the RhD gene with a fluorescence-based polymerase-chain-reaction (PCR) test sensitive enough to detect the RhD gene in a single cell. Fetal RhD status was determined directly by serologic analysis of cord blood or PCR analysis of amniotic fluid. RESULTS: Among the 57 RhD-negative women, 12 were in their first trimester of pregnancy, 30 were in their second trimester, and 15 were in their third trimester. Thirty-nine fetuses were RhD-positive, and 18 were RhD-negative. In the samples obtained from women in their second or third trimester of pregnancy, the results of RhD PCR analysis of maternal plasma DNA were completely concordant with the results of serologic analysis. Among the maternal plasma samples collected in the first trimester, 2 contained no RhD DNA, but the fetuses were RhD-positive; the results in the other 10 samples were concordant (7 were RhD-positive, and 3 RhD-negative). CONCLUSIONS: Noninvasive fetal RhD genotyping can be performed rapidly and reliably with the use of maternal plasma beginning in the second trimester of pregnancy.

Blood Grouping and Crossmatching↗
PubMed1998

Molecular cytogenetic delineation of the critical deleted region in the 5q- syndrome.

The 5q- syndrome is a distinct type of myelodysplastic syndrome (MDS) characterised by refractory anaemia, morphological abnormalities of megakaryocytes, and del(5q) as the sole cytogenetic abnormality. In contrast to patients with therapy-related MDS with 5q deletions, 5q- syndrome patients have a favourable prognosis and a low rate of transformation to acute leukaemia. We have previously delineated a common deleted region of 5.6 Mb between the gene for fibroblast growth factor acidic (FGF1) and the subunit of interleukin 12 (IL12B) in two patients with 5q- syndrome and small deletions, del(5)(q31q33). The present study used fluorescence in situ hybridisation (FISH) analysis of these and a third 5q- syndrome patient with a small deletion, del(5)(q33q34), to refine further the critical deleted region. This resulted in the narrowing of the common deleted region within 5q31.3-5q33 to approximately 3 Mb, flanked by the adrenergic receptor beta 2 (ADRB2) and IL/2B genes. The common region of loss in these three 5q- syndrome patients includes the macrophage colony-stimulating factor-1 receptor (CSF1R), secreted protein, acidic, cysteine-rich (SPARC), and glutamate receptor (GR1A1) genes. This 5q- syndrome critical region is telomeric to and distinct from the other critical regions on 5q associated with MDS and acute myeloid leukaemia.

Adult↗
PubMed1998

Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis.

We have developed a real-time quantitative PCR assay to measure the concentration of fetal DNA in maternal plasma and serum. Our results show that fetal DNA is present in high concentrations in maternal plasma, reaching a mean of 25.4 genome equivalents/ml (range 3.3-69. 4) in early pregnancy and 292.2 genome equivalents/ml (range 76. 9-769) in late pregnancy. These concentrations correspond to 3.4% (range 0.39%-11.9%) and 6.2% (range 2.33%-11.4%) of the total plasma DNA in early and late pregnancy, respectively. Sequential follow-up study of women who conceived by in vitro fertilization shows that fetal DNA can be detected in maternal serum as early as the 7th wk of gestation and that it then increases in concentration as pregnancy progresses. These data suggest that fetal DNA can be readily detected in maternal plasma and serum and may be a valuable source of material for noninvasive prenatal diagnosis.

DNA↗