Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing.

Abstract

Adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, we synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNAs showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These changes do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency.

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene, and expression of inactive mutant enzyme protein in E. coli

“…Deletion of exon 2 has been described previously by Strauss and coworkers in a Dutch patient, who died during an attack caused by MCAD deficiency ). The deletions of exons 2, 5 and 8 can only be explained by mis-splicing, which has been observed for other genes (Akeson et al 1987; Ohno and Suzuki 1988). The relevance for MCAD deficiency is unclear, but it might be speculated either that mutation of residue 985 destabilizes the splice complexes, giving rise to exon skipping, or that the accumu-lated metabolites affect the precursor mRNA splice processing.…”

Evidence for a complex regulatory array in the first intron of the human adenosine deaminase gene.

“…A single major cap site is used in T-lymphoid cells (Valerio et al 1985;Akeson et al 1987), and a fragment of DNA that extends 135 bp, 5' of the cap site functions as a promoter in transient expression assays in a variety of cell types (Valerio et al 1985, this study). In this laboratory, Wiginton et al (1986) has determined the sequence of 32 kb of transcribed and 3.9 kb of 5'-flanking DNA encom-passing the human gene, and Lattier et al (1989) have recently shown that at least some of the variations in expression of the enzyme are the result of variations in transcription of the gene.…”

Hot spot mutations in adenosine deaminase deficiency.

“…Sequencing of cDNA revealed a CG to TG transition at base 631, resulting in replacement of basic arginine by cysteine at codon 211, again consistent with expression of mutant abnormally acidic ADA. This codon is also the site ofmutation in an ADA-SCID, where the corresponding CG to CA hot spot transition results in replacement of arginine by histidine (30). Sequencing of cDNA also confirmed the T320 to C transition.…”

“…Therefore, if one considers only mutations resulting in partial ADA deficiency, there is an unusually high frequency of hot spot mutations (5/6). This high frequency may be true for all mutations at the ADA locus since 6 of 10 null single base-pair mutations are hot spot mutations [6 previously described (23,(30)(31)(32), 1 reported here, and 3 unpublished findings]. One of these 6 hot spot mutations is truly recurrent, having appeared on two different chromosomal backgrounds (ref.…”