GM02825

GM02825 LCL from B-Lymphocyte

Description:

SEVERE COMBINED IMMUNODEFICIENCY, AUTOSOMAL RECESSIVE, T CELL-NEGATIVE, B CELL-NEGATIVE, NK CELL-NEGATIVE, DUE TO ADENOSINE DEAMINASE DEFICIENCY
ADENOSINE DEAMINASE; ADA

Affected:

Yes

Sex:

Female

Age:

4 MO (At Sampling)

Overview

Repository

NIGMS Human Genetic Cell Repository

Subcollection

Heritable Diseases

Class

Disorders of Nucleotide and Nucleic Acid Metabolism

Biopsy Source

Peripheral vein

Cell Type

B-Lymphocyte

Tissue Type

Blood

Transformant

Epstein-Barr Virus

Sample Source

LCL from B-Lymphocyte

Race

White

Family Member

Relation to Proband

proband

Confirmation

Clinical summary/Case history

Species

Homo sapiens

Common Name

Human

Remarks

Clinically affected; lymphoblastoid cell line contains less than 5% of normal ADA activity and immunoreactive protein; absent IgM and IgA; weak MLC response; absent T cells; no detectable ADA activity in erythrocytes; normal ADA mRNA level but cells contain mRNAs of abnormal size; normal ADA mRNAs and ADA mRNAs missing exon 4 are present in approximately equal abundance; donor subject is a compund heterozygote: one allele has a C>T transition at nucleotide 1081 in exon 11 of the ADA gene [1081C>T] resulting in a substitution of valine for alanine at codon 329 [Ala329Val(A329V)] and a second allele has an A>G transition in the 3-prime splice site of intron 3 of the ADA gene resulting in a deletion of exon 4 from mature RNA; same donor as GM02824 fibroblast.

Characterizations

IDENTIFICATION OF SPECIES OF ORIGIN

Species of Origin Confirmed by Nucleoside Phosphorylase, Glucose-6-Phosphate Dehydrogenase, and Lactate Dehydrogenase Isoenzyme Electrophoresis

adenosine deaminase

Wiginton et al (J Biol Chem 257:3211-3217 1982) observed that this cell culture contained <5% of normal ADA activity and immunoreactive protein. Adrian et al (Hum Genet 68:169-172 1984) reported that this ADA-deficient cell culture had normal relative levels of ADA mRNA. This ADA mRNA was found to be of abnormal size (Adrian et al Mol Cell Biol 4:1712-1717 1984). S1 nuclease mapping revealed two ADA mRNA bands of equal intensity. One of these bands was full-length ADA mRNA and the other band was shorter lacking exon 4. Upon analysis of cDNA synthesized from this culture ADA mRNA revealed a point mutation in the allele which produced the full-length mRNA. This mutation at base 1081 (GCG>GTG) changes amino acid 329 from alanine to valine. Several of the ADA cDNA clones extended 5 prime of the major initiation start site indicating multiple start sights for ADA transcription. Blot hybridization of genomic DNA eliminated the possibility of a large deletion in the gene that included exon 4. Isolation and sequence analysis of cDNAs corresponding to this allele showed no mutations within the remaining exons that would alter the amino acid sequence. Akeson et al (Proc Natl Acad Sci USA 84:5947-5951 1987) concluded that it seemed likely that mutation(s) within intron 3 and/or intron 4 caused deletion of exon 4 during the splicing process. The deletion of exon 4 does not alter the translational reading frame but it is likely that any protein produced would be rapidly degraded. Akeson et al (J Biol Chem 263:16291-16296 1988) reported that sequence analysis of polymerase chain reaction-amplified DNA from this culture showed a single base mutation of A to G within the invariant bases of the 3 prime splice site of intron 3 that could account for the missplicing of exon 4. This mutation occured in base 24971 of the ADA gene. Insertion of the mutant ADA cDNA from this cell line into an expression vector and testing in a transient expression system revealed that ADA mRNAs transcribed from the mutant vector failed to produce significant levels of enzymatically active ADA while ADA mRNA transcribed from a vector with the normal ADA cDNA did produce high levels of enzymatically active ADA. These authors also noted that the same mutation in amino acid no. 329 (Ala to Val) resulting from a point mutation in base no. 1081 (C to T) was also found in another ADA-deficient cell line (GM02756). EC Number: 3.5.4.4; <5% activity.

GENE MAPPING & DOSAGE STUDIES - Y CHROMOSOME

PCR analysis of DNA from this cell culture gave a negative result with a primer for Yq11, DYS227.

Gene

ADA

Chromosomal Location

20q13.11

Allelic Variant 1

608958.0006; SEVERE COMBINED IMMUNODEFICIENCY, AUTOSOMAL RECESSIVE, T CELL-NEGATIVE, B CELL-NEGATIVE, NK CELL-NEGATIVE, DUE TO ADENOSINE DEAMINASE DEFICIENCY

Identified Mutation

ALA329VAL; In GM02756, Akeson et al. [Proc Natl Acad Sci U S A 84: 5947 (1987)] demonstrated 2 different mutant alleles: one was ala329 to val; the other was a 632G-A transition in the ADA gene, resulting in the replacement of the arginine residue by histidine at codon 211 (102700.0004). In addition, Akeson et al. [Proc Natl Acad Sci U S A 84: 5947 (1987)] and [J Biol Chem 263:16291 (1988)] found that cell line GM02825A was a genetic compound. One allele had an ala329-to-val change (102700.0006); the other allele had a point mutation from A to G in the 3-prime splice site of intron 3 (102700.0017), resulting in elimination of exon 4 from the mature mRNA.

Gene

ADA

Chromosomal Location

20q13.11

Allelic Variant 2

608958.0017; SEVERE COMBINED IMMUNODEFICIENCY, AUTOSOMAL RECESSIVE, T CELL-NEGATIVE, B CELL-NEGATIVE, NK CELL-NEGATIVE, DUE TO ADENOSINE DEAMINASE DEFICIENCY

Identified Mutation

IVS3AS, A>G, -2, EX4DEL; Akeson et al. [Proc Natl Acad Sci U S A 84: 5947 (1987)] and [J Biol Chem 263:16291 (1988)] found that cell line GM02825 was a genetic compound. One allele had an ala39-to-val change (102700.0006); the other allele had a point mutation from A to G in the 3-prime splice site of intron 3 (102700.0017), resulting in elimination of exon 4 from the mature mRNA.

Phenotypic Data

Remarks

Publications

Hirschhorn R, Identification of two new missense mutations (R156C and S291L) in two ADA- SCID patients unusual for response to therapy with partial exchange transfusions. Hum Mutat1:166-8 1992

PubMed ID: 1284479

Hirschhorn R, Ellenbogen A, Tzall SHirschhorn, Five missense mutations at the adenosine deaminase locus (ADA) detected by altered restriction fragments and their frequency in ADA--patients with severe combined immunodeficiency (ADA-SCID). Am J Hum Genet42:201-7 1992

PubMed ID: 1346349

Hirschhorn R, Chakravarti V, Puck J, Douglas SD, Homozygosity for a newly identified missense mutation in a patient with very severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID). Am J Hum Genet49:878-85 1991

PubMed ID: 1680289

Markert ML, Norby-Slycord C, Ward FE, A high proportion of ADA point mutations associated with a specific alanine-to-valine substitution. Am J Hum Genet45:354-61 1989

PubMed ID: 2773932

Tzall S, Ellenbogen A, Eng F, Hirschhorn R, Identification and characterization of nine RFLPs at the adenosine deaminase (ADA) locus. Am J Hum Genet44:864-75 1989

PubMed ID: 2567118

Akeson AL, Wiginton DA, Dusing MR, States JC, Hutton JJ, Mutant human adenosine deaminase alleles and their expression by transfection into fibroblasts. J Biol Chem263:16291-6 1988

PubMed ID: 3182793

Akeson AL, Wiginton DA, States JC, Perme CM, Dusing MR, Hutton JJ, Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing. Proc Natl Acad Sci U S A84:5947-51 1987

PubMed ID: 3475710

Berkvens TM, Gerritsen EJ, Oldenburg M, Breukel C, Wijnen JT, van Ormondt H, Vossen JM, van der Eb AJ, Meera Khan P, Severe combined immune deficiency due to a homozygous 3.2-kb deletion spanning the promoter and first exon of the adenosine deaminase gene. Nucleic Acids Res15:9365-78 1987

PubMed ID: 3684597

Valerio D, Dekker BM, Duyvesteyn MG, van der Voorn L, Berkvens TM, van Ormondt H, van der Eb AJ, One adenosine deaminase allele in a patient with severe combined immunodeficiency contains a point mutation abolishing enzyme activity. EMBO J5:113-9 1986

PubMed ID: 3007108

Adrian GS, Wiginton DA, Hutton JJ, Characterization of normal and mutant adenosine deaminase messenger RNAs by translation and hybridization to a cDNA probe. Hum Genet68:169-72 1984

PubMed ID: 6548726

Adrian GS, Wiginton DA, Hutton JJ, Structure of adenosine deaminase mRNAs from normal and adenosine deaminase-deficient human cell lines. Mol Cell Biol4:1712-7 1984

PubMed ID: 6208479

Daddona PE, Shewach DS, Kelley WN, Argos P, Markham AF, Orkin SH, Human adenosine deaminase. cDNA and complete primary amino acid sequence. J Biol Chem259:12101-6 1984

PubMed ID: 6090454

Wiginton DA, Hutton JJ, Immunoreactive protein in adenosine deaminase deficient human lymphoblast cell lines. J Biol Chem257:3211-7 1982

PubMed ID: 6977542

Hirschhorn R, Roegner V, Jenkins T, Seaman C, Piomelli S, Borkowsky W, Erythrocyte adenosine deaminase deficiency without immunodeficiency. Evidence for an unstable mutant enzyme. J Clin Invest64:1130-9 1979

PubMed ID: 479373