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1.
Am J Hum Genet ; 81(5): 987-94, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17924340

ABSTRACT

Congenital heart defects (CHDs) are among the most common birth defects in humans (incidence 8-10 per 1,000 live births). Although their etiology is often poorly understood, most are considered to arise from multifactorial influences, including environmental and genetic components, as well as from less common syndromic forms. We hypothesized that disturbances in left-right patterning could contribute to the pathogenesis of selected cardiac defects by interfering with the extrinsic cues leading to the proper looping and vessel remodeling of the normally asymmetrically developed heart and vessels. Here, we show that heterozygous loss-of-function mutations in the human GDF1 gene contribute to cardiac defects ranging from tetralogy of Fallot to transposition of the great arteries and that decreased TGF- beta signaling provides a framework for understanding their pathogenesis. These findings implicate perturbations of the TGF- beta signaling pathway in the causation of a major subclass of human CHDs.


Subject(s)
Genetic Predisposition to Disease , Heart Defects, Congenital/genetics , Intercellular Signaling Peptides and Proteins/genetics , Mutation/genetics , Amino Acid Sequence , Animals , DNA Mutational Analysis , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Growth Differentiation Factor 1 , Humans , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Molecular Sequence Data , Phenotype , Protein Structure, Secondary , RNA, Messenger/genetics , RNA, Messenger/metabolism , Zebrafish/embryology , Zebrafish/genetics
2.
N Engl J Med ; 336(5): 317-23, 1997 Jan 30.
Article in English | MEDLINE | ID: mdl-9011783

ABSTRACT

BACKGROUND: Complete remission of B-precursor acute lymphoblastic leukemia (ALL) has traditionally been defined as the near absence of lymphoblasts in a light-microscopical examination of stained bone marrow smears, but a patient in remission may still harbor up to 10(10) leukemia cells. We investigated whether there is a relation between the outcome of treatment and submicroscopic evidence of residual disease. METHODS: We conducted a prospective study of patients during a first clinical remission using a quantitative polymerase-chain-reaction (PCR) assay capable of detecting 1 viable leukemia cell among 200,000 normal marrow mononuclear cells and a clonogenic blast-colony assay. Bone marrow specimens from 24 children were sequentially evaluated during a five-year period, and the results were compared with the clinical outcome. RESULTS: Seven patients relapsed and 17 remained in remission 2 to 35 months after the completion of treatment. The levels of residual leukemia-cell DNA in the two groups were significantly different (P<0.001; 95 percent confidence interval for the difference in the mean log-transformed ratio of leukemia-cell DNA to normal bone marrow-cell DNA, 0.38 to 1.28). Autoregression analyses identified trends for individual patients that were associated with relapse. Despite continued remission in 17 patients, evidence of residual leukemia was detected by PCR in 15 and by both PCR and blast-colony assays in 7. CONCLUSIONS: Molecular signs of residual leukemia can persist up to 35 months after the cessation of chemotherapy in children with ALL in remission. This suggests that eradication of all leukemia cells may not be a prerequisite for cure.


Subject(s)
Bone Marrow/pathology , Neoplastic Stem Cells/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Child , Clone Cells , DNA, Neoplasm/analysis , Disease-Free Survival , Humans , Neoplasm, Residual , Polymerase Chain Reaction , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Prospective Studies , Recurrence , Remission Induction , Tumor Stem Cell Assay
3.
J Cell Physiol ; 166(3): 618-30, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8600166

ABSTRACT

Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukemia (ALL) is a malignant disorder characterized by a poor prognosis. In recent years hematopoietic growth factors have been used to recruit myeloid leukemia blasts into the proliferative phase of the cell cycle and as supportive agents, both with cytotoxic regimens and in the setting of bone marrow transplantation. This approach prompted us to investigate whether myeloid growth factors have a role in Ph1 positive ALL. To do this, we utilized two newly established Ph1-positive cell lines, Z-119 and Z-181. Both lines have L2 morphology, ultrastructural characteristics of lymphoblasts and typical B-lineage surface markers identical to those observed in the two Ph1-positive ALL patients from whom they were derived. In addition, a single rearranged immunoglobulin heavy-chain gene (JH) band was found in both cell lines by Southern blot analysis, confirming B-cell clonality. Cytogenetic analysis of the two lines revealed t(9;22). Polymerase chain reaction (PCR) amplified cDNA from both Z-119 and Z-181 cells revealed an e1--a2 BCR-ABL junction, and p190BCR-ABL protein was detected in them by the immune complex kinase assay. Both cell lines produce interleukin (IL)-1 beta, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF), but neither IL-1 beta, G-CSF, their corresponding antibodies and inhibitory molecules, nor GM-CSF, affected the cell lines' growth. However, GM-CSF neutralizing antibodies inhibited Z-181 but not Z-119 colony formation in a dose-dependent fashion by up to 77% and addition of GM-SCF reversed this inhibitory effect. Receptor studies with radiolabeled GM-CSF demonstrated specific binding to Z-181 but not to Z-119 cells, and Scatchard analysis revealed that Z-181 cells express high-affinity GM-CSF receptors. Furthermore, PCR analysis showed that Z-181 but not Z-119 bears the transcript for the GM-CSF receptor. Finally, studies using PH1-positive ALL patients' marrow cells revealed similar data. In 3 of 8 samples we detected significant concentrations of GM-CSF (7.5-13 pg/2 x 10(7) cells) and in 2 of 3 cases GM-CSF significantly stimulated Ph1-positive ALL colony proliferation. These data suggest that Ph1-positive ALL cells may produce GM-CSF, express GM-CSF receptors and thus show a proliferative response to this cytokine.


Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/physiology , Philadelphia Chromosome , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Adult , B-Lymphocytes/chemistry , Base Sequence , Biomarkers, Tumor/analysis , Bone Marrow/chemistry , Bone Marrow/pathology , Cell Division , Chromosomes, Human, Pair 22 , Clone Cells , Female , Fusion Proteins, bcr-abl/analysis , Fusion Proteins, bcr-abl/genetics , Gene Rearrangement, B-Lymphocyte, Heavy Chain , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Growth Substances/biosynthesis , Humans , Karyotyping , Male , Molecular Sequence Data , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , RNA, Messenger/analysis , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Tumor Cells, Cultured
4.
Leukemia ; 9(2): 321-8, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7869771

ABSTRACT

The detection of residual leukemia cells in the bone marrow of patients during morphologic remission has been greatly facilitated by use of the polymerase chain reaction (PCR) to amplify leukemia-specific sequences. While the current PCR strategies for estimating the amount of residual leukemia claim a detection sensitivity of one leukemia cell amongst 10(5) or 10(6) normal cells, a rigorous assessment of the relative error associated with these techniques has not been presented. We have developed a method of estimating the amount of residual leukemia in remission marrows that is analogous to the limiting dilution assays used to determine the frequency of immunocompetent cells in a responder cell population. Using this method we measured the fraction of all-or-none (i.e. positive or negative) reactions of the PCR amplification of the leukemia-specific IgH gene rearrangement in replicate samples of serial dilutions of DNA obtained from diagnostic bone marrow specimens from 15 children with B-precursor acute lymphoblastic leukemia (ALL). A sigmoid curve representing the fraction of positive PCR reactions at a given dilution of leukemia DNA was found to be the best fit to the data. The narrowness of the log-linear region of this curve prevents the direct application of the analysis methodology that has previously been described for limiting dilution assays. However, the residual leukemia burden during morphological remission in these 15 patients and in two additional patients who experienced relapse could be estimated by the described dilution analysis method using the best-fit equation. Furthermore, the data generated for diagnostic, remission and relapse marrow samples exhibited a small interspecimen variation. The results suggest that this method can reliably estimate residual leukemia over a range of five orders of magnitude. Although the PCR reaction appears to be one of the most sensitive methods for detecting residual leukemia, all techniques based on this procedure, including our own, must exhibit limitations inherent to the amplification process. Our estimates or relative error suggest that a realistic limit for the PCR estimation of residual leukemia lies in the range of one leukemia cell per 10(5) normal cells. The suggested method is rapid, technically simple and relatively inexpensive. Furthermore, the principles that it is based upon can be applied to any PCR-based strategy.


Subject(s)
Bone Marrow Examination/methods , Gene Rearrangement, B-Lymphocyte, Heavy Chain , Polymerase Chain Reaction , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Child , DNA, Neoplasm/genetics , Genes, Immunoglobulin , Humans , Immunoglobulin Heavy Chains/genetics , Neoplasm, Residual , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Reproducibility of Results , Sensitivity and Specificity
5.
Lancet ; 343(8901): 858-9, 1994 Apr 02.
Article in English | MEDLINE | ID: mdl-7908106
6.
Leukemia ; 8(1): 46-52, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8289497

ABSTRACT

No effective therapy is available for the majority of the 30-40% of children with acute lymphoblastic leukemia (ALL) who relapse. Since the morphologically undetectable, or occult, leukemia cells that persist during remission originate from the clone present at diagnosis, may also have both the capability to sustain the disease and to give rise to relapse, we are evaluating a method of identifying them. We have combined, for the first time, an ALL blast colony assay (BCA) and the polymerase chain reaction (PCR) to isolate residual leukemia cells in remission bone marrow aspirate specimens from eight patients with B-precursor ALL during early continuation therapy. We found colony-forming leukemia cells with in vitro self-renewal capability that survived chemotherapy for 15 months after diagnosis in all sequential specimens from these patients. To verify the leukemic nature of these cells their DNA was amplified by PCR and the product directly sequenced. In every case, the VHDJH sequence observed at diagnosis was found. None of the patients relapsed during this early phase of their treatment, consistent with the observation that patients with B-precursor ALL experience recurrence late in their course. Since it is possible that some of these persistent leukemia cells belong to the leukemia progenitor cell population that sustains the disease, the study of them could provide the means to determine the mechanisms of relapse.


Subject(s)
Neoplastic Stem Cells/pathology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Adolescent , Base Sequence , Bone Marrow/physiology , Bone Marrow Cells , Child , Child, Preschool , Clone Cells , DNA, Neoplasm/genetics , Female , Humans , Male , Molecular Sequence Data , Neoplastic Stem Cells/physiology , Polymerase Chain Reaction , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Remission Induction , T-Lymphocytes/pathology
7.
Eur J Biochem ; 205(2): 443-9, 1992 Apr 15.
Article in English | MEDLINE | ID: mdl-1572350

ABSTRACT

Type IX collagen is composed of three genetically distinct polypeptides that contain several collagenous and non-collagenous domains. The alpha 2(IX) chain also contains a covalently bound glycosaminoglycan side chain. Type IX collagen is located on the surface of collagen fibrils of both hyaline cartilage and vitreous humor, such that one of the collagenous domains (COL3) projects from the surface of the fibril in a periodic manner. We have cloned and sequenced a full-length cDNA for the chicken alpha 3(IX) collagen chain from a cartilage cDNA library. Together with the sequence of the alpha 1(IX) and alpha 2(IX) chains, this completes the primary structure of type IX collagen for one species. These sequences will be useful to better understand the mechanism of triple-helix formation in type IX collagen and the nature of type II and type IX collagen interactions in fibril formation.


Subject(s)
Collagen/genetics , Amino Acid Sequence , Animals , Base Sequence , Cartilage/physiology , Chick Embryo , Chickens , Cloning, Molecular/methods , DNA/genetics , Macromolecular Substances , Molecular Sequence Data , Oligodeoxyribonucleotides , Oligonucleotides, Antisense , Polymerase Chain Reaction , Sequence Homology, Nucleic Acid
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