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R Hirschhorn

Publications and source records attributed to R Hirschhorn.

At least 37 records · Page 2Linked to original sources
PubMed1996

Spontaneous in vivo reversion to normal of an inherited mutation in a patient with adenosine deaminase deficiency.

Somatic mosaicism in genetic disease generally results from a de novo deleterious mutation during embryogenesis. We now describe a somatic mosaicism due to the unusual mechanism of in vivo reversion to normal of an inherited mutation. The propositus was an adenosine deaminase-deficient (ADA-) child with progressive clinical improvement and unexpectedly mild biochemical and immunologic abnormalities. Mosaicism due to reversion was evidenced by absence of a maternally transmitted deleterious mutation in 13/15 authenticated B cell lines and in 17% of single alleles cloned from blood DNA, despite retention of a maternal 'private' ADA polymorphism linked to the mutation. Establishment of significant somatic mosaicism following reversion to normal could modify any disorder in which revertant cells have a selective advantage.

Adenosine Deaminase↗
PubMed1996

Identification of an E689K substitution as the molecular basis of the human acid alpha-glucosidase type 4 allozyme (GAA*4).

We have identified the molecular basis of the GAA*4 allozyme as a G to A transition at nt2065 which predicts the substitution of glutamic acid by lysine at codon 689 (E689K). The conclusion that this change represents the molecular basis of the GAA*4 allozyme is based on 1) presence of the G2065A in homozygosity in a known GAA*4 homozygote, 2) transient expression studies showing normal enzyme activity expressed by cDNA containing the G2065A transition and 3) isoelectric focusing studies showing a more cathodal pattern for the expressed product as compared to the common GAA*1, analogous to the patterns seen in normal and known GAA*4 lymphoid cells.

Asian People↗
PubMed1995

Identification of C1q as the heat-labile serum cofactor required for immune complexes to stimulate endothelial expression of the adhesion molecules E-selectin and intercellular and vascular cell adhesion molecules 1.

To examine the role of complement components as regulators of the expression of endothelial adhesive molecules in response to immune complexes (ICs), we determined whether ICs stimulate both endothelial adhesiveness for leukocytes and expression of E-selectin and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1). We found that ICs [bovine serum albumin (BSA)-anti-BSA] stimulated endothelial cell adhesiveness for added leukocytes in the presence of complement-sufficient normal human serum (NHS) but not in the presence of heat-inactivated serum (HIS) or in tissue culture medium alone. Depletion of complement component C3 or C8 from serum did not prevent enhanced endothelial adhesiveness stimulated by ICs. In contrast, depletion of complement component C1q markedly inhibited IC-stimulated endothelial adhesiveness for leukocytes. When the heat-labile complement component C1q was added to HIS, the capacity of ICs to stimulate endothelial adhesiveness for leukocytes was completely restored. Further evidence for the possible role of C1q in mediating the effect of ICs on endothelial cells was the discovery of the presence of the 100- to 126-kDa C1q-binding protein on the surface of endothelial cells (by cytofluorography) and of message for the 33-kDa C1q receptor in resting endothelial cells (by reverse transcription-PCR). Inhibition of protein synthesis by cycloheximide blocked endothelial adhesiveness for leukocytes stimulated by either interleukin 1 or ICs in the presence of NHS. After stimulation with ICs in the presence of NHS, endothelial cells expressed increased numbers of adhesion molecules (E-selectin, ICAM-1, and VCAM-1). Endothelial expression of adhesion molecules mediated, at least in part, endothelial adhesiveness for leukocytes, since leukocyte adhesion was blocked by monoclonal antibodies directed against E-selectin. These studies show that ICs stimulate endothelial cells to express adhesive proteins for leukocytes in the presence of a heat-labile serum factor. That factor appears to be C1q.

Antigen-Antibody Complex↗
PubMed1995

Evidence of molecular heterogeneity for generalised glycogenosis between and within breeds of cattle.

Northern analyses revealed normal levels of acidic alpha-glucosidase mRNA in cultured fibroblasts from a Shorthorn calf affected with glycogenosis but a gross deficiency in an affected Brahman calf. Analyses of acidic alpha-glucosidase activity, relative to that of other lysosomal enzymes, in blood mononuclear cells revealed greater variation within and between Brahman herds than Shorthorn herds. A Msp1 restriction fragment length polymorphism associated with glycogenosis in Brahmans was not found in Shorthorns. These results are considered in relation to molecular heterogeneity for AAG deficiency in cattle and its implications for disease control programs.

Animals↗
PubMed1994

Homozygosity for a missense mutation (G20R) associated with neonatal onset adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID).

Mutations at the adenosine deaminase (ADA) locus can result in varying degrees of immunodeficiency, including rapidly fulminant severe combined immunodeficiency (SCID) as well as a slowly progressive immunodeficiency not diagnosed until later in childhood. Genetic heterogeneity is a factor in the clinical heterogeneity. We have now identified, by direct sequencing of PCR-amplified genomic DNA, a G to A transition at a CpG dinucleotide predicting a glycine to arginine substitution at codon 20 (G20R). The mutation, in homozygosity, was associated with neonatal-onset rapidly fatal SCID. Consistent with homozygosity, the child was derived from a small isolated inbred community in Newfoundland. The mutation abolishes a site for the restriction enzyme BamHI and can be simply detected by agarose gel electrophoresis following amplification of exon 2 from genomic DNA and digestion with BamHI. The majority of ADA missense mutations can now be detected by similar amplification and enzyme digestion. We demonstrated that the G20R mutation is deleterious since introduction of the mutation into a normal ADA minigene abolished enzyme activity, as determined by transient expression in monkey kidney (Cos) cells. The amino acid substitution occurs in an area of the molecule conserved from Escherichia coli to man and that, as shown by crystallographic analysis, is involved in the binding of Zn2+ at the catalytic site. Although the mutation is in a CpG dinucleotide, known "hotspots" for G to A transitions, it was not found in a series of 43 additional ADA- chromosomes. Identification of mutations in additional ADA- patients with immunodeficiency of varying severity should further define the role that genotype plays in determining the extent of immunologic dysfunction.

Adenosine Deaminase↗
PubMed1994

Aberrant splicing in adult onset glycogen storage disease type II (GSDII): molecular identification of an IVS1 (-13T-->G) mutation in a majority of patients and a novel IVS10 (+1GT-->CT) mutation.

Two newly identified splice site mutations (IVS1 -13T-->G and IVS10 +1GT-->CT) were found in a patient with adult onset of the autosomal recessive disorder glycogen storage disease type II (GSDII). The IVS1 -13T-->G transversion in the acceptor splice site was found on one allele in over two thirds of adult onset GSDII patients studied (28/41), but was not seen in 58 normal or 12 infantile onset GSDII chromosomes. Molecular analysis of cDNA from the index patient and four additional, ethnically different, individuals carrying the IVS1 -13T-->G transversion showed splicing out of the first coding exon as well as rare utilization of a cryptic splice site in the exon. An IVS10 +1GT-->CT transversion, unique to the index patient, was detected on the second chromosome. The IVS10 +1GT-->CT results in splicing out of exon 10 including part of the enzyme catalytic site. Additionally, a large deletion encompassing exon 18, previously described in four unrelated patients, was also detected in three unrelated adult GSDII patients, two of whom carried the IVS1 -13T-->G transversion. The frequency of the IVS1 splice site mutation suggests that detection of this mutation could potentially aid in the diagnosis of the phenotypically variable syndrome of adult onset GSDII. The finding that the -13T-->G mutation is a very common mutation in adult onset GSDII patients of varying ethnic and racial backgrounds, suggests that it is either an ancient mutation or confers a selective advantage. Although to our knowledge these are the first splice site mutations to be reported for GSDII, additional splice site mutations are likely and could provide the basis for later onset disease in GSDII.

Adult↗
PubMed1994

A de novo 13 nt deletion, a newly identified C647W missense mutation and a deletion of exon 18 in infantile onset glycogen storage disease type II (GSDII).

We identified the presumably rare event of de novo mutation in an autosomal recessive disorder, glycogen storage disease type II (GSDII). GSDII results from inherited deficiency of acid alpha-glucosidase (acid maltase) and both the expressed and structural gene (designated GAA) have been isolated. The mutation was a deletion of 13 nt of coding sequence (delta nt 1456-1468) on the paternally derived allele of the proband. The delta nt 1456-1468 results in a reading frameshift and a premature termination signal upstream of the enzyme catalytic site. Paternity was confirmed by presence of two downstream, uncommon amino acid substitutions (E689K, W746C) in both proband and father and by comparison of nine short tandem repeats. The maternal allele carried a newly identified deleterious C647W missense mutation in a highly conserved area of the protein. The C647W mutation was also found in a second unrelated proband, heteroallelic with a deletion extending from IVS17 to IVS18.

Alleles↗
PubMed1994

Crossover of placebo patients to intravenous immunoglobulin confirms efficacy for prophylaxis of bacterial infections and reduction of hospitalizations in human immunodeficiency virus-infected children. The National Institute of Child Health and Human Development Intravenous Immunoglobulin Clinical Trial Study Group.

After completion of a placebo-controlled trial of intravenous immunoglobulin (IVIG) infection prophylaxis, patients were offered open label IVIG and optional participation in a follow-up study. The purpose of the follow-up study was to evaluate the IVIG effect in original placebo recipients and longevity of IVIG benefit in original IVIG recipients. Of 212 human immunodeficiency virus-infected children on study at trial closure, 148 (67 of 98 (68%) placebo and 81 of 114 (71%) IVIG patients) received open label IVIG for a mean of 16 months. When open label IVIG was begun, 45% were receiving trimethoprim-sulfamethoxazole prophylaxis for Pneumocystis carinii pneumonia (43% of placebo and 47% of IVIG patients) and 54% were receiving zidovudine (55% of placebo and 53% of IVIG patients). In patients who received placebo during the original study, the rate of serious bacterial infections was significantly lower after change to open label IVIG (estimated 15.8 fewer episodes/100 patient years; 95% confidence interval, 3.2 to 28.5; P = 0.014). Similar findings were observed for minor bacterial infections (estimated 61.2 fewer/100 patient years; 95% confidence interval, 29.2 to 93.3; P < 0.001) and hospitalizations (estimated 43.7 fewer/100 patient years; 95% confidence interval, 27.7 to 59.6; P < 0.001). Decreases were observed whether or not trimethoprim-sulfamethoxazole prophylaxis was being given at the time open label IVIG was begun. In patients who received IVIG during the original study, no significant difference was seen in infections or hospitalizations after change to open label IVIG.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS-Related Opportunistic Infections↗
PubMed1994

Somatic mosaicism for a newly identified splice-site mutation in a patient with adenosine deaminase-deficient immunodeficiency and spontaneous clinical recovery.

Absent or severely reduced adenosine deaminase (ADA) activity produces inherited immunodeficiency of varying severity, with defects of both cellular and humoral immunity. We report somatic mosaicism as the basis for a delayed presentation and unusual course of a currently healthy young adult receiving no therapy. He was diagnosed at age 2 1/2 years because of life-threatening pneumonia, recurrent infections, failure of normal growth, and lymphopenia, but he retained significant cellular immune function. A fibroblast cell line and a B cell line, established at diagnosis, lacked ADA activity and were heteroallelic for splice-donor-site mutation in IVS 1 (+1GT-->CT) and a missense mutation (Arg101Gln). All clones (17/17) isolated from the B cell mRNA carried the missense mutation, indicating that the allele with the splice-site mutation produced unstable mRNA. In striking contrast, a B cell line established at age 16 years expressed 50% of normal ADA; 50% of ADA mRNA had normal sequence, and 50% had the missense mutation. Genomic DNA contained the missense mutation but not the splice-site mutation. All three cell lines were identical for multiple polymorphic markers and the presence of a Y chromosome. In vivo somatic mosaicism was demonstrated in genomic DNA from peripheral blood cells obtained at 16 years of age, in that less than half the DNA carried the splice-site mutation (P < .002, vs. original B cell line). Consistent with mosaicism, erythrocyte content of the toxic metabolite deoxyATP was only minimally elevated. Somatic mosaicism could have arisen either by somatic mutation or by reversion at the site of mutation. Selection in vivo for ADA normal hematopoietic cells may have played a role in the return to normal health, in the absence of therapy.

Adenosine Deaminase↗
PubMed1994

An Asp8Asn substitution results in the adenosine deaminase (ADA) genetic polymorphism (ADA 2 allozyme): occurrence on different chromosomal backgrounds and apparent intragenic crossover.

We have now determined the molecular genetic basis for the common biochemical polymorphism at the adenosine deaminase (ADA) locus. The ADA*2 allele contains a G to A transition at nt22 (relative to the ATG) that results in substitution of asparagine for aspartic acid at codon 8 (Asp8Asn). Introduction of the nucleotide substitution into an ADA 1 cDNA and transfection into monkey kidney (Cos) cells confirmed that the mutation resulted in expression of an enzyme that comigrated with the naturally occurring ADA 2 allozyme. The substitution of neutral asparagine for anionic aspartic acid is consistent with the more cathodal electrophoretic migration of ADA 2 as compared with ADA 1. The nucleotide substitution was found on at least two different genetic backgrounds, suggesting independent recurrence of the mutation. Consistent with independent recurrence, the G to A transition is at a CpG dinucleotide and represents a type of mutation that occurs with high frequency. We have also unexpectedly identified a probable intragenic crossover in the very large first intron that is rich in repetitive DNA sequences.

Adenosine Deaminase↗
PubMed1993

Severe combined immunodeficiency of reduced severity due to homozygosity for an adenosine deaminase missense mutation (Arg253Pro).

Genetic deficiency of adenosine deaminase (ADA) results in varying degrees of immunodeficiency, including neonatal onset severe combined immunodeficiency (ADA- SCID) and milder, later onset immunodeficiency. We have determined the molecular basis of disease in a child from a consanguineous mating with ADA- SCID of clinically and biochemically reduced severity, diagnosed at 15 months of age and characterized by retention of more immunologic function than is typical of the fulminant neonatal onset type. The course was notable for an early predominance of bacterial infections and eosinophilia. In contrast to its absence in most ADA- SCIDs, residual ADA activity (1-2% of normal) could be detected in EBV-transformed B cells. Consistent with the increased residual ADA, excretion of the substrate deoxyadenosine and accumulation of the toxic metabolite deoxyATP were less than seen in ADA- SCID patients with fulminant disease. Sequence analysis of cDNA revealed a G853C transversion, predicting a substitution of proline for arginine at codon 253 (Arg253Pro). The parents were heterozygous and the child was homozygous for the mutation, as shown by sequence analysis of amplified genomic DNA. Transient expression of mutant cDNA in Cos cells revealed an electrophoretically abnormal, more negatively charged ADA with 1-2% of normal activity. These observations are consistent with replacement of positively charged arginine by proline, the lower accumulation of toxic metabolites, and the milder phenotype. By contrast, transient expression of a Gly216Arg mutant cDNA, associated, when homozygous, with neonatal onset ADA-SCID, did not reveal ADA activity. Mutations such as Arg253Pro, which retain residual activity of monomeric ADA, should be dominant for ameliorating the phenotype in patients carrying two different allelic mutations. Identification of additional similar mutations may be significant in evaluating the goals for and efficacy of current trials of gene and gene product replacement.

Adenosine Deaminase↗
PubMed1993

Overview of biochemical abnormalities and molecular genetics of adenosine deaminase deficiency.

Approximately 20 years ago, Giblett and coworkers serendipitously discovered that in some patients with the syndrome of severe combined immunodeficiency, the disease is due to an inherited deficiency of the enzyme adenosine deaminase (ADA). This then led to the discovery that inherited deficiency of the next enzyme in the same pathway for purine salvage, purine nucleoside phosphorylase, results in a profound defect in cell-mediated immunity. These two disorders, sometimes termed "purinergic immunodeficiency disorders," were the first of the inherited immunodeficiency disorders in which the specific molecular basis was determined. Although both are rare diseases, they are of importance for several reasons. First, they are among the few inherited disorders of which some children can be cured by a single treatment; second, they are ideally suited for gene therapy; and third, the pathologic mechanisms can tell us more about the nature of the immune system and have already allowed development of chemotherapy for some malignancies of the immune system. Knowledge of the specific defects has also facilitated diagnosis, counseling of families, and development of new approaches to therapy. This article focuses on ADA deficiency, briefly reviews the clinical and biochemical findings to provide a background for the next two articles in this supplement issue, and details work in progress with respect to several aspects of the specific molecular defects in different patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Deaminase↗
PubMed1992

Novel deletion and a new missense mutation (Glu 217 Lys) at the catalytic site in two adenosine deaminase alleles of a patient with neonatal onset adenosine deaminase- severe combined immunodeficiency.

Mutations at the adenosine deaminase (ADA) locus result in a spectrum of disorders, encompassing a fulminant neonatal onset severe combined immunodeficiency (SCID) and childhood onset immunodeficiency, as well as apparently normal immune function. The extent of accumulation of the toxic metabolite, deoxyATP, correlates directly with severity of disease. We have now determined the mutations on both alleles of a child with fulminant, neonatal onset ADA- SCID and accumulation of extremely high concentrations of deoxyATP. The genotype was consistent with the severely affected phenotype. One allele carried a large deletion that arose by non-homologous recombination and included the first five exons and promoter region. The second allele carried a missense mutation (G649A) resulting in replacement of Glu217, an amino acid involved in the catalytic site, by Lys and predicting a major alteration in charge. Expression of the mutant cDNA in Cos cells confirmed that the mutation abolished enzyme activity. We have previously reported that a missense mutation at the preceding codon is similarly associated with neonatal onset ADA- SCID and accumulation of extremely high deoxyATP. These findings suggest that genotype-phenotype correlations may be apparent for ADA- SCID, despite the role that random variation in exposure to environmental pathogens may play in the initial phenotype. Such genotype-phenotype correlations may be important to consider in evaluating results of ongoing trials of "gene" and enzyme replacement therapy.

Adenosine Deaminase↗