Mutations and polymorphisms in the human ornithine transcarbamylase gene

Deletions of variable size involving one or more exons, 29 different missense, nonsense, or frameshift mutations, and three polymorphisms have been found in patients with ornithine transcarbamylase (OTC) deficiency. Most of the deletions and mutations were found in patients with severe disease manifested clinically as acute neonatal hyperammonemia. A small number of mutations or somatic mosaicism for deletions were found in males with “late onset” disease and in heterozygous females who were symptomatic. Approximately 10–15% of all molecular alterations associated with OTC defi ciency are large deletions involving all or part of the OTC gene with or without contiguous genes on the short arm of the X chromosome. Approximately 10% of all point mutations involve the CpG dinucleotide of codon 141 with a CGA→CAA transition producing a deleterious Arg→Gln substitu tion in position 109 of the mature enzyme and causing the elimination of a TaqI recognition site. The majority of the remaining mutations in the OTC gene are unique to the affected family and are usually not found in unrelated patients. To date, two mutations have been described in the sequence of the “leader” peptide, 23 mutations have been found in the coding sequence of the “mature” enzyme, and four mutations have been discovered in splicing recognition sites. Approximately 20 single base polymorphisms have been postulated to exist by comparing two reported OTC gene sequences; six of these substitutions cause amino acid changes of which three have been confirmed in patients. Of the known point mutations, 27 are single base substitutions: 17 missense, 6 nonsense, 4 splice site, and the remaining 2 are single base deletions. © 1993 Wiley-Liss, Inc.     

Kinds and locations of mutations arising spontaneously in the coding region of theHPRT gene of finite-life-span diploid human fibroblasts

Spontaneous thioguanine-resistant mutants were derived from populations of finite-life-span, diploid human fibroblasts by means of a fluctuation analysis. cDNA was prepared from mutantHPRT mRNA and amplified by the polymerase chain reaction, and the sequence of the product was analyzed. Exon deletions, which very likely arose from mutations in the intron splice site consensus sequences, were found in 10 of the 37 mutants examined (27% of the total). Among the 28 mutations in the coding sequence, base pair substitutions predominated (89%). With the exception of one base pair involved in a tandem mutation, all base pair substitutions resulted in alterations in the predicted amino acid sequence of the protein. In addition there were three frameshift mutations, consisting of the deletion of one or two base pairs. Although mutations occurred throughout the coding sequence, 50% (14/28) were found in the 5 portion of exon 3.     

Molecular nature of spontaneous mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in chinese hamster ovary cells

The hypoxanthine-guanine phosphoribosyltransferase (hprt) locus has been widely used as a selectable genetic marker for studies of mammalian cell mutagenesis. We report here the spontaneous mutation spectrum at the hprt locus in 64 independently isolated mutants of Chinese hamster ovary (CHO) cells. All nine hprt exons were simultaneously analyzed via multiplex polymerase chain reaction (PCR) for rapid detection of gene deletions or insertions. Structural point mutations were identified by direct sequence analysis of the PCR amplified cDNA. The molecular nature of RNA splicing errors and insertions was analyzed by solid-phase direct exon sequencing. Single base substitutions were found in 24 mutants (38%), of which 21 were missense and 3 were nonsense mutations. Transversions were about twice as frequent as transitions. Fifteen mutants (23%) had deletions involving either intragenic small fragments (2), single exons (9), or multiple exons (4). The majority of deletion breakpoints (71%) were located in regions surrounding exons 4, 5, and 6. RNA splicing mutations were observed in 15 mutants (23%) and affected exons 3–8; most (6/15) resulted in the loss of exon 7. Two insertion mutants, one with a 209 bp insert in exon 4 and the other with a 88 bp insert accompanied by a 24 bp deletion in exon 6, represent novel mutations reported for the first time in spontaneous mutants of the mammalian hprt gene. © 1995 Wiley-Liss, Inc.     

Reported in vivo splice-site mutations in the factor IX gene: severity of splicing defects and a hypothesis for predicting deleterious splice donor mutations

Small consensus sequences have been defined for RNA splicing, but questions about splicing in humans remain unanswered. Analysis of germline mutations in the factor IX gene offers a highly advantageous system for studying the mutational process in humans. In a sample of 860 families with hemophilia B, 9% of independent mutations are likely to disrupt splicing as their primary mode of action. This includes 26 splicing mutations reported herein. When combined with the factor IX splice mutations reported by others, at least 104 independent mutations have been observed, 80 of which are single base substitutions within the splice donor and splice acceptor consensus sequences. After analysis of these mutations, the following inferences emerge: (1) the susceptibility of a splice donor sequence to deleterious mutation depends on the degree of similarity with the donor consensus sequence, suggesting a simple "5-6 hypothesis" for predicting deleterious vs. neutral mutations; (2) the great majority of mutations that disrupt the splice donor or splice acceptor sequences result in at least a 100-fold decrement in factor IX coagulant activity, indicating that the mutations at these sites generally function as an on/off switch; (3) mutations that create cryptic splice junctions or that shorten but do not interrupt the polypyrimidine tract in the splice acceptor sequence can reduce splicing by a variable amount; and (4) there are thousands of potential donor-acceptor consensus sequence combinations in the 38-kb factor IX gene region apparently not reduced by evolutionary selective pressure, presenting an apparent paradox; i.e., mutations in the donor and acceptor consensus sequences at intron/exon splice junctions can dramatically alter normal splicing, yet, appropriately spaced, good matches to the consensus sequences do not predispose to significant amounts of alternative splicing.     

Mutations causing defective splicing in the human hprt gene.

Ten intron mutations and one exon mutation giving rise to defective splicing in the human gene for hypoxanthine phosphoribosyl transferase (hprt) in T-lymphocytes have been characterized. The splicing mutants were detected by PCR amplification of hprt cDNA and direct sequencing. Nine of the mutants showed skipping of whole exons or parts of exons in the cDNA, one mutant had an inclusion of an intron sequence into the cDNA, and one mutant showed both inclusion of an intron sequence and skipping of exons as well as a normal cDNA. Genomic PCR and direct sequencing of the splice sites involved showed one deletion of three base pairs and 10 different single base alterations to be responsible for these splice alterations. One mutation in the last base pair of exon 6 causing skipping of the entire exon 6 was found, whereas an identical mutation in the last base pair of exon 2 caused no aberrant splicing. It was also found that a deletion mutation in the pyrimidine rich stretch of the acceptor site of intron 7 caused skipping of the entire exon 8, whereas a base substitution in the last base of intron 7 caused exclusion of only the first 21 base pairs of exon 8 as a result of the activation of a cryptic acceptor site in exon 8. The results show that many different types of mutations at several different sites can cause splicing errors in the hprt gene and that the sequence differences between the splice sites influence the possible spectrum of mutations in each site.     

Molecular analysis of mutations affecting hprt mRNA splicing in human T-lymphocytes in vivo.

Molecular analysis of hypoxanthine-guanine phosphoribosyltransferase (hprt) cDNA from 6-thioguanine-resistant T-lymphocytes cloned from smoking and non-smoking adult donors showed that 35% of these mutants were defective in splicing of hprt mRNA. Among a set of 42 hprt splice mutants, we observed i) complete loss of one or more exons, ii) partial loss of one exon, or iii) inclusion of part of an intron sequence between adjacent exons. Loss of exon 4 was significantly more frequent than of the other exons, suggesting that the sequences that regulate splicing of this exon are either larger than those of the other exons or especially prone to mutation. In order to identify the molecular nature of DNA alterations causing aberrant splicing of hprt mRNA, 17 splice mutants were analyzed in more detail by sequencing the genomic regions flanking the mis-spliced exon. Base pair substitutions or small deletions causing defective splicing were either detected in exon sequences or in splice site consensus sequences of introns. Furthermore, genomic deletions encompassing entire exons were found. In some mutants, the alteration responsible for incorrect splicing could not be identified, suggesting that the target sequence for splice mutations is larger than merely the splice junctions. Molecular characterization of hprt splice mutations will lead to the identification of specific sequences regulating splicing of hprt mRNA and will reveal whether the mutational spectrum in splice mutants is similar to that found in the hprt coding region.     

DNA sequence analysis of hprt mutations in lymphocytes from Sprague-Dawley rats treated with 7,12-dimethylbenz[a]anthracene

Treatment of female Sprague-Dawley rats with the potent mammary gland carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) results in the formation of DNA adducts with dG and dA and in the induction of 6-thioguanine-resistant (TG′) lymphocyte mutants. In this study, we have examined the types of mutations induced in TG′ lymphocytes from DMBA-treated rats. DNA from 263 TG′ lymphocyte clones was screened for mutations in exons 2, 3, and 8 of the hprt gene by polymerase chain reaction (PCR) amplification of the exons followed by heteroduplex analysis using denaturing gradient-gel electrophoresis. Twenty-five of the clones produced heteroduplexes in exon 2, 35 produced heteroduplexes in exon 3, and 36 produced heteroduplexes in exon 8. Direct sequence analysis of the heteroduplexes revealed 96 mutations, and at least 74 of these mutations were produced independently. Eighty-five of the total mutations were simple base pair (bp) substitutions, with A → T and G → T transversions being the predominant types. Seven mutations were deletions, three were complex bp substitutions, and one was an insertion. The results suggest that the types of mutations produced by DMBA in rat lymphocytes are specific to the DNA adducts produced by this compound. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Utilisation of a cryptic non-canonical donor splice site of the gene encoding PARAFIBROMIN is associated with familial isolated primary hyperparathyroidism

More than 99% of all splice sites conform to consensus sequences that usually include the invariant dinucleotides gt and ag at the 5' and 3' ends of the introns, respectively. We report on the utilisation of a non-consensus (non-canonical) donor splice site within exon 1 of the HRPT2 gene in familial isolated primary hyperparathyroidism (FIHP). HRPT2 mutations are more frequently associated with the hyperparathyroidism-jaw tumour syndrome (HPT-JT). Patients with FIHP were identified to have a donor splice site mutation, IVS1+1 g-->a, and the consequences of this for RNA processing were investigated. The mutant mRNA lacked 30 bp and DNA sequence analysis revealed this to result from utilisation of an alternative cryptic non-canonical donor splice site (gaatgt) in exon 1 together with the normally occurring acceptor splice site in intron 1. Translation of this mutant mRNA predicted the in-frame loss of 10 amino acids in the encoded protein, termed PARAFIBROMIN. Thus, these FIHP patients are utilising a ga-ag splice site pair, which until recently was considered to be incompatible with splicing but is now known to occur as a rare (<0.02%) normal splicing variant.     

Determinants of the 3' splice site for self-splicing of the Tetrahymena pre-rRNA

Tetrahymena preribosomal RNA undergoes self-splicing in vitro. The structural components involved in recognition of the 5' splice site have been identified, but the mechanism by which the 3' splice site is recognized is not established. To identify some components of 3'splice site recognition, we have generated mutations near the 3' splice site and determined their effects on self-splicing. Alteration of the 3'-terminal guanosine of the intervening sequence (IVS), a conserved nucleotide in group I IVSs, almost eliminated 3' splice site activity; the IVS-3' exon splicing intermediate accumulated, and exon ligation was extremely slow. These mutations do not result in recruitment of cryptic 3' splice sites, in contrast to mutations that affect the 5' splice site. Alteration of the cytidine preceding the 3'-terminal guanosine or of the first two nucleotides of the 3' exon had similar but less severe effects on exon ligation. Most of the mutants showed some reduction (less than threefold) in GTP addition at the 5' splice site. A mutation that placed a new guanosine residue just upstream from the 3'-terminal guanosine misspliced to produce ligated exons with one extra nucleotide between the 5' and 3' exons. We conclude that multiple nucleotides, located both at the 3' end of the IVS and in the 3' exon, are required for 3' splice site recognition.     

Prediction of Mutant mRNA Splice Isoforms by Information Theory‐Based Exon Definition

Mutations that affect mRNA splicing often produce multiple mRNA isoforms, resulting in complex molecular phenotypes. Definition of an exon and its inclusion in mature mRNA relies on joint recognition of both acceptor and donor splice sites. This study predicts cryptic and exon‐skipping isoforms in mRNA produced by splicing mutations from the combined information contents (R_i, which measures binding‐site strength, in bits) and distribution of the splice sites defining these exons. The total information content of an exon (R_i,total) is the sum of the R_i values of its acceptor and donor splice sites, adjusted for the self‐information of the distance separating these sites, that is, the gap surprisal. Differences between total information contents of an exon (ΔR_i,total) are predictive of the relative abundance of these exons in distinct processed mRNAs. Constraints on splice site and exon selection are used to eliminate nonconforming and poorly expressed isoforms. Molecular phenotypes are computed by the Automated Splice Site and Exon Definition Analysis ( http://splice.uwo.ca ) server. Predictions of splicing mutations were highly concordant (85.2%; n = 61) with published expression data. In silico exon definition analysis will contribute to streamlining assessment of abnormal and normal splice isoforms resulting from mutations.     

Identification of mutations in the Bruton's tyrosine kinase gene,including a novel genomic rearrangements resulting in large deletion,in Korean X-linked agammaglobulinemia patients

Mutations in the Bruton's tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA). We identified BTK mutations in six patients with presumed XLA from unrelated Korean families. Four out of six mutations were novel: two missense mutations (P565T, C154Y), a point mutation in a splicing donor site (IVS11+1G>A), and a large deletion (a 6.1-kb deletion including BTK exons 11–18). The large deletion, identified by long-distance PCR, revealed Alu-Alu mediated recombination extended from an Alu sequence in intron 10 to another Alu sequence in intron 18, spanning a distance of 6.1 kb. The two known mutations consisted of one missense (G462D) mutation, and a point mutation in a splicing acceptor site (IVS7−9A>G). This study suggests that large genomic rearrangements involving Alu repeats are few but an important component of the spectrum of BTK mutations.