Biomedical subjects
Publications and source records attributed to M L Markert.
Explore the source record for details and available documents.
From May 1992 to March 1993, 50 infants with severe combined immunodeficiency (SCID) were given bone marrow transplants at Duke University Medical Center. None received chemotherapy for conditioning or for graft-versus-host disease (GVHD) prophylaxis. Forty-one received haploidentical parental marrow depleted of T cells by soybean lectin and sheep red blood cell resetting, and nine received HLA-identical marrow. Forty (80%) survived from 1 week to almost 11 years posttransplantation, including nine of nine (100%) HLA-identical marrow recipients and 31 of 41 haploidentical recipients. T-cell function was present within 2 weeks after transplantation of unfractionated HLA-identical marrow, but not until 3 to 4 months after T-cell-depleted haploidentical marrow stem cells. All 37 patients who are more than 4 months posttransplantation have good T-cell function, and all but one have 100% donor T cells. B-cell function developed slowly or not at all in some recipients of haploidentical marrow. Fourteen (four HLA-identical and 10 haploidentical recipients) have some donor B cells; 19 patients are receiving intravenous immune globulin (IVIG) therapy.
We report a defect in splicing of precursor messenger RNA (pre-mRNA) resulting from a naturally occurring mutation of the gene encoding purine nucleoside phosphorylase (PNP) in a patient with PNP-deficient severe combined immunodeficiency. This defects results from a G to T transversion at the terminal nucleotide of exon 2 within the 5' splice site of intron 2 and causes skipping of exon 2 during processing of PNP pre-mRNA. Translation of the misspliced mRNA results in a reading frameshift at the exon 1-exon 3 junction. The predicted polypeptide encoded by the aberrant mRNA is severely truncated, terminating at 31 amino acids. Only 4 residues at the NH2 terminus of the polypeptide correspond to PNP amino acids. Otherwise the translation product of the misspliced mRNA differs completely from PNP in amino acid sequence and has no PNP activity. The finding of exon skipping in PNP is the first report of a splicing defect resulting in PNP-deficient severe combined immunodeficiency. Analysis of the genomic context of the G-1 to T mutation of the 5' splice site lends support for the exon definition model of pre-mRNA splicing and contributes to the understanding of splice site selection.
Purine nucleoside phosphorylase (PNP) deficiency is an inherited autosomal recessive disorder resulting in severe combined immunodeficiency. The purpose of this study was to determine the molecular defects responsible for PNP deficiency in one such patient. The patient's PNP cDNA was amplified by PCR and sequenced. Point mutations leading to amino acid substitutions were found in both alleles. One point mutation led to a Ser-to-Gly substitution at amino acid 51 and was common to both alleles. In addition, an Asp-to-Gly substitution at amino acid 128 and an Arg-to-Pro substitution at amino acid 234 were found in the maternal and paternal alleles, respectively. In order to prove that these mutations were responsible for the disease state, each of the three mutations was constructed separately by site-directed mutagenesis of the normal PNP cDNA, and each was transiently expressed in COS cells. Lysates from cells transfected with the allele carrying the substitution at amino acid 51 retained both function and immunoreactivity. Lysates from cells transfected with PNP alleles carrying a substitution at either amino acid 128 or amino acid 234 contained immunoreactive material but had no detectable human PNP activity. In summary, molecular analysis of this patient identified point mutations within the PNP gene which are responsible for the enzyme deficiency.
Purine nucleoside phosphorylase (PNP) deficiency is a rare inherited disease accounting for approximately 4% of patients with severe combined immunodeficiency. Thirty-three patients have been reported. PNP-deficient patients suffer from recurrent infections, usually beginning in the first year of life. Two thirds of patients have evidence of neurologic disorders. Findings range from spasticity to developmental delay, to mental retardation. One third of patients develop autoimmune disease. The most common manifestation of this is autoimmune hemolytic anemia. Idiopathic thrombocytopenic purpura and systemic lupus erythematosis have also been reported. Patients usually present with infections but approximately one fourth have come to medical care initially for neurological problems. In PNP deficiency, T- and B-cell immunity are affected. T-cell function may be profoundly deficient, may be normal at birth and then decrease with time, or may fluctuate repeatedly between low and normal. B-cell function can be normal but is deficient in approximately one third of patients. PNP protein is a trimer of approximately 90,000 daltons. It is found in most tissues of the body but is at highest levels in lymphoid tissues. This tissue distribution explains why the lymphoid system is predominantly affected in PNP deficiency. Many mechanisms have been proposed to explain the metabolic toxicity in PNP deficiency. The elevated dGTP found in PNP deficiency is thought to inhibit ribonucleotide reductase and, thus, impede cell division. Depressed GTP levels may correlate with neurologic dysfunction. The gene for PNP has been cloned; it is located on the long arm of chromosome 14. Studies of a mutant PNP gene isolated from one patient showed that a point mutation resulting in an amino acid substitution was responsible for PNP deficiency. PNP deficiency has a grave prognosis. No patient has reached the third decade of life. Twenty-nine of the 33 reported patients have died from their disease. Prenatal diagnosis is currently available. Many different therapies have been utilized for PNP deficiency including bone marrow transplantation, red cell transfusions, and supplementation of the diet with purines and pyrimidines. None of these therapies has been consistently successful. In light of the poor prognosis for PNP deficiency, bone marrow transplantation should be considered for all patients. In the future, improved forms of therapy such as gene therapy may become available.
In 15%-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The overall goal of our research has been to identify the precise molecular defects in patients with ADA-deficient SCID. In this study, we focused on a patient whom we found to have normal sized ADA mRNA by Northern analysis and an intact ADA structural gene by Southern analysis. By cloning and sequencing this patient's ADA cDNA, we found a C-to-T point mutation in exon 11. This resulted in the amino acid substitution of a valine for an alanine at position 329 of the ADA protein. Sequence analysis revealed that this mutation created a new BalI restriction site. Using Southern analyses, we were able to directly screen individuals to determine the frequency of this mutation. By combining data on eight families followed at our institution with data on five other families reported in the literature, we established that five of 13 patients (seven of 22 alleles) with known or suspected point mutations have this defect. This mutation was found to be associated with three different ADA haplotypes. This argues against a founder effect and suggests that the mutation is very old. In summary, a conservative amino acid substitution is found in a high proportion of patients with ADA deficiency; this can easily be detected by Southern analysis.
In 15-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The goal of this study was to determine the precise molecular defect in a patient with ADA-deficient SCID whom we previously have shown to have a total absence of ADA mRNA and a structural alteration of the ADA gene. By detailed Southern analysis, we now have determined that the structural alteration is a deletion of approximately 3.3 kb, which included exon 1 and the promoter region of the ADA gene. DNA sequence analysis demonstrates that the deletion created a novel, complete Alu repeat by homologous recombination between two existing Alu repeats that flanked the deletion. The 26-bp recombination joint in the Alu sequence includes the 10-bp "B" sequence homologous to the RNA polymerase III promoter. This is the first example of homologous recombination involving the B sequence in Alu repeats. Similar recombination events have been identified involving Alu repeats in which the recombination joint was located between the A and B sequences of the polymerase III split promoter. The nonrandom location of these events suggests that these segments may be hot spots for recombination.
A patient with adenosine deaminase-deficient severe combined immunodeficiency is described whose defect is secondary to deletion of a portion of the ADA structural gene. In Southern analyses, DNA from this patient does not hybridize to a genomic probe that includes the 3' end of exon 1. This implies that both his parents are heterozygous for deletions of exon 1 sequences. Consistent with this finding, the patient has no detectable adenosine deaminase mRNA by Northern analysis. This is the first report of a deletion mutation as the cause of adenosine deaminase deficiency.
We treated two children who had adenosine deaminase deficiency and severe combined immunodeficiency disease by injecting bovine adenosine deaminase modified by conjugation with polyethylene glycol. The modified enzyme was rapidly absorbed after intramuscular injection and had a half-life in plasma of 48 to 72 hours. Weekly doses of approximately 15 U per kilogram of body weight maintained plasma adenosine deaminase activity at two to three times the level of erythrocyte adenosine deaminase activity in normal subjects. The principal biochemical consequences of adenosine deaminase deficiency were almost completely reversed. In erythrocytes, adenosine nucleotides increased and deoxyadenosine nucleotides decreased to less than 0.5 percent of total adenine nucleotides. The activity of S-adenosylhomocysteine hydrolase, which is inactivated by deoxyadenosine, increased to normal in red cells and nucleated marrow cells. Neither toxic effects nor hypersensitivity reactions were observed. In vitro tests of the cellular immune function of each patient showed marked improvement, along with an increase in circulating T lymphocytes. Clinical improvement was indicated by absence of infection and resumption of weight gain. We conclude that from the standpoints of efficacy, convenience, and safety, polyethylene glycol-modified adenosine deaminase is preferable to red-cell transfusion as a treatment for adenosine deaminase deficiency. Patients with other inherited metabolic diseases in which accumulated metabolites equilibrate with plasma could benefit from treatment with the appropriate polyethylene glycol-modified enzyme.
The courses of six patients with adenosine deaminase (ADA) and two with purine nucleoside phosphorylase (PNP) deficiencies were evaluated before and after therapy. The heterogeneity of immunologic and clinical parameters was striking in each enzyme deficiency. In both PNP and ADA deficiency, some patients had very low immunoglobulin levels, while others had normal levels. T-cell function was always low in patients with ADA deficiency. In the two patients with PNP deficiency, contrary to the classical descriptions of this disorder, T-cell function fluctuated with time. Five ADA-deficient patients were treated with irradiated normal red-cell transfusions as a form of enzyme replacement and showed no lasting benefit. Three of the ADA-deficient patients and one of the PNP-deficient patients were given transplants of haploidentical parental bone marrow stem cells without pretransplant immunosuppression. In the PNP-deficient patient, chimerism has not been documented on enzymatic testing. One ADA-deficient patient has demonstrated long-term engraftment with good B- and T-cell function. Haploidentical bone marrow transplantation is currently the preferred therapy for enzyme-deficient patients with absent T-cell function who do not have an HLA-identical donor, as it may result in a lasting reconstitution of immune function. In those patients with unsatisfactory responses to transplantation, however, specific enzyme replacement or gene therapy may be considered in the future.
Explore the source record for details and available documents.
Recent advances in the prevention of graft-vs-host disease (GVHD) have allowed the use of haploidentical bone marrow cells for correction of lethal genetic defects of the immune system. Sequential analyses of blood lymphocyte phenotypes and functions were done before and after transplantation of haploidentical marrow stem cells into 17 infants with severe primary T cell deficiencies. The marrow was depleted of post-thymic T cells and most other mature marrow cells by soy lectin agglutination and sheep erythrocyte rosetting. The studies were performed to define the time course and extent of appearance of immune function, and to identify factors leading to resistance to engraftment. No pretransplant immunosuppression was used. T cell function was detected between 34 and 287 days after transplantation, but a sharp rise usually occurred between 84 and 115 days, and normal function was reached between 113 and 210 days. Fifteen of the patients are alive from 6 to 41 mo post-transplantation, 12 have improved or have normal T lymphocyte function, and nine have proven T cell chimerism. Increased immunoglobulins of several isotypes have been noted in 11 patients and specific antibodies in seven patients, although B cell chimerism has been detected in only one patient. B cell function required 2 to 2.5 yr for normalization. No GVHD occurred in 14 patients, and the other three had only transient mild skin rashes. Two patients died of viral infections. Failure to engraft was correlated with some pre-transplant lymphocyte responses to mitogens and allogeneic cells (three cases), but not with the presence of pre-transplant natural killer cell function (five cases) nor with the presence of purine salvage pathway enzyme deficiencies (four cases). The latter, however, was associated with poor lymphoid function in two patients. These studies indicate that the thymic microenvironment of most infants with severe combined immunodeficiency disease is capable of differentiating donor stem cells to mature and functioning T lymphocytes which can cooperate with apparently normal host B cells for antibody production.
Four HLA-DR-homozygous cell lines were examined in detail to compare alpha and beta subunits precipitated by chimpanzee antisera and alloantisera directed against HLA-DR, MB, or MT determinants. For the HLA-DR5-homozygous cell line Sweig, chimpanzee antisera directed against MT2 and against the HLA-DR5 specificity yielded identical patterns. Similar findings for MT and MB vs HLA-DR were obtained for the cell lines LG14 (HLA-DR3-homozygous), PBur (HLA-DR7-homozygous), and Eld (HLA-DRw6-homozygous). Therefore, on some cell lines MT and/or MB specificities appear to be detectable only on the same or electrophoretically identical alpha-beta complexes as HLA-DR determinants.
Human B cell alloantigens were studied by two-dimensional gel electrophoresis. The heavy (alpha) and light (beta) chains precipitated by xenoantisera were compared on B lymphoblastoid cell lines of different HLA-DR types. In accordance with the results of other investigators, we observed considerable electrophoretic variability of beta subunits. Significant electrophoretic differences between alpha subunits isolated from different cells were also observed through the use of a pH 5 to 7 gradient in the isoelectric focusing dimension. HLA-DR allelic products from a heterozygous line exhibited differences in beta subunit electrophoretic mobility but apparent identity of alpha subunits.
The B-cell alloantigens of an HLA-DR3-homozygous, MB2-homozygous, MT2-positive lymphoblastoid cell line were studied by two-dimensional gel electrophoresis. Analysis of gel patterns suggested assignment of the HLA-DR3 determinant to the larger (35,000-dalton) subunit (alpha) of the B-cell alloantigen. MB2 was found to be either a determinant on the small (27,000 dalton) subunit (beta) or a crossreactive determinant(s) on the HLA-DR3 alpha subunit and an additional alpha subunit. MT2 was found to be a determinant on Ia antigen-like molecules distinct from those carrying the MB and HLA-DR determinants. The results are consistent with the existence in the major histocompatibility complex of at least three loci encoding B-cell alloantigens.