Nonsense and missense mutations in hemophilia A: estimate of the relative mutation rate at CG dinucleotides.

Hemophilia A is an X-linked disease of coagulation caused by deficiency of factor VIII. Using cloned cDNA and synthetic oligonucleotide probes, we have now screened 240 patients and found CG-to-TG transitions in an exon in nine. We have previously reported four of these patients; and here we report the remaining five, all of whom were severely affected. In one patient a TaqI site was lost in exon 23, and in the other four it was lost in exon 24. The novel exon 23 mutation is a CG-to-TG substitution at the codon for amino acid residue 2166, producing a nonsense codon in place of the normal codon for arginine. Similarly, the exon 24 mutations are also generated by CG-to-TG transitions, either on the sense strand producing nonsense mutations or on the antisense strand producing missense mutations (Arg to Gln) at position 2228. The novel missense mutations are the first such mutations observed in association with severe hemophilia A. These results provide further evidence that recurrent mutations are not uncommon in hemophilia A, and they also allow us to estimate that the extent of hypermutability of CG dinucleotides is 10-20 times greater than the average mutation rate for hemophilia A.     

The molecular genetics of haemophilia A: screening for point mutations in the factor VIII gene using the restriction enzyme TaqI

Summary A combination of Southern blotting and the analysis of polymerase chain reaction (PCR) amplified DNA fragments was used to screen the factor VIII genes of 527 haemophilia A patients for point mutations within TaqI restriction sites. Since this directed search strategy yielded only four gene lesions, it was concluded that its efficacy is less than that originally predicted. One novel point mutation was however found in a moderately severe haemophiliac; a CGA (Arg) to CTA (Leu) transversion at codon 2209, an evolutionarily conserved residue in the C2 domain of the factor VIII protein. The remaining three detected lesions, CGA (Arg)TGA (Term) transitions at codons 2116, 2147 and 2307, respectively, have been reported before and are consistent with recurrent mutation at these hypermutable sites. A number of TaqI restriction site polymorphisms/rare variants were also noted. These variants appear to be population-specific but are nevertheless potentially useful in individual cases as intragenic markers for carrier detection and antenatal diagnosis.     

Recurrent nonsense mutations at arginine residues cause severe hemophilia B in unrelated hemophiliacs

Summary Direct sequencing of the regions of the factor IX gene of likely functional significance was performed in four patients with severe hemophilia B. In two of the individuals, a transition at the dinucleotide CpG caused a nonsense mutation at arginine 333. In the other two individuals, a transition at CpG caused a nonsense mutation at arginine 29. Since these patients are all unrelated, as shown by differing alleles of the TaqI polymorphism in intron four or extensive nonoverlapping pedigrees, the mutations arose independently. In addition, the origin of one arginine 333 mutation in one family has been traced to the germline of the maternal grandfather. The frequent occurrence of mutations at arginine codons that contain the sequence CGN can be explained by the dramatic elevation of transitions at CpG. As a result, approximately one in four individuals with hemophilia B is expected to have a mutation at arginine and nonsense mutations at one of six arginine residues should be common causes of severe hemophilia.     

Characterization of mutations in the factor VIII gene by direct sequencing of amplified genomic DNA

In order to search for mutations resulting in hemophilia A that are not detectable by restriction analysis, three regions of the factor VIII gene were chosen for direct sequence analysis. Short segments of genomic DNA of 127 unrelated patients with hemophilia A were amplified by polymerase chain reaction. A total of 136,017 nucleotides were sequenced, and four mutations leading to the disease were found: a frameshift at codon 360 due to deletion of two nucleotides (GA), a nonsense codon 1705 due to a C----T transition, and two missense codons at positions 1699 and 1708. The first missense mutation (A----T) results in a Tyr----Phe substitution at a putative von Willebrand factor binding site. The second results in an Arg----Cys substitution at a thrombin cleavage site. In addition, we identified three rare sequence variants: a silent C----T transition at codon 34 which does not result in an amino acid change, a G----C change at codon 345 (Val----Leu), and an A----G change at the third nucleotide of intron 14. Direct sequence analysis of amplified DNA is a powerful but labor-intensive method of identifying mutations in large genes such as the human factor VIII gene.     

Identification of splicing mutations of the last nucleotides of exons, a nonsense mutation, and a missense mutation of the XPAC gene as causes of group A xeroderma pigmentosum.

Four mutations of the XPAC gene were identified as molecular bases of different UV-sensitive subgroups of xeroderma pigmentosum (XP) group A. One was a G to C transversion at the last nucleotide of exon 4 in GM1630/GM2062, a little less hypersensitive subgroup than the most sensitive XP2OS/XP12RO. The second mutation was a G to A transition at the last nucleotide of exon 3 in GM2033/GM2090, an intermediate subgroup. Both mutations caused almost complete inactivation of the canonical 5' splice donor site and aberrant RNA splicing. The third mutation was a nucleotide transition altering the Arg-211 codon (CGA) to a nonsense codon (TGA) in another allele of GM2062. The fourth mutation was a nucleotide transversion altering the His-244 codon (CAT) to an Arg codon (CGT) in XP8LO, an intermediate subgroup. Our results strongly suggest that the clinical heterogeneity in XP-A is due to different mutations in the XPAC gene.     

Recurrent nonsense mutations within the type VII collagen gene in patients with severe recessive dystrophic epidermolysis bullosa.

The generalized mutilating form of recessive dystrophic epidermolysis bullosa (i.e., the Hallopeau-Siemens type; HS-RDEB) is a life-threatening disease characterized by extreme mucocutaneous fragility associated with absent or markedly altered anchoring fibrils (AF). Recently, we reported linkage between HS-RDEB and the type VII collagen gene (COL7A1), which encodes the major component of AF. In this study, we investigated 52 unrelated HS-RDEB patients and 2 patients with RDEB inversa for the presence, at CpG dinucleotides, of mutations changing CGA arginine codons to premature stop codons TGA within the COL7A1 gene. Eight exons containing 10 CGA codons located in the amino-terminal domain of the COL7A1 gene were studied. Mutation analysis was performed using denaturing gradient gel electrophoresis of PCR-amplified genomic fragments. Direct sequencing of PCR-amplified products with altered electrophoretic mobility led to the characterization of three premature stop codons, each in a single COL7A1 allele, in four patients. Two patients (one affected with HS-RDEB and the other with RDEB inversa) have the same C-to-T transition at arginine codon 109. Two other HS-RDEB patients have a C-to-T transition at arginine 1213 and 1216, respectively. These nonsense mutations predict the truncation of approximately 56%-92% of the polypeptide, including the collagenous and the noncollagenous NC-2 domains. On the basis of linkage analysis, which showed no evidence for locus heterogeneity in RDEB, it is expected that these patients are compound heterozygotes and have additional mutations on the other COL7A1 allele, leading to impaired AF formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

CG dinucleotide transitions in the factor IX gene account for about half of the point mutations in hemophilia B patients: a Seattle series

Summary Hemophilia B is due to multiple molecular defects in the factor IX gene. Over 80% of mutations are single base substitutions. By amplification and direct sequencing, 51 single base substitutions were found in the transcribed sequence of the factor IX genes of patients from 50 distinct families with hemophilia B. These include 30 mutations in 29 families not previously reported by us; of these, 12 are novel, i.e., not previously published in other series. Of the 51 substitutions in our overall series 23 (45%) occurred as C-to-T or G-to-A transitions at 11 sites within CG dinucleotides. It is estimated that CG transitions occur from one to two orders of magnitude more frequently than mutations in nucleotides that are not within a CG pair. More than one family had identical defects for 6 of the CG mutations. At 4 of these sites, most patients had different haplotypes compatible with distinct mutations. Non-CG-type mutations occurred thoughout the coding regions with only one mutation in more than one family. The latter included 7 families with a 397 Ile-to-Thr defect that all share a rare haplotype, suggesting a common ancestor.     

Three nonsense mutations responsible for group A xeroderma pigmentosum.

The molecular basis of xeroderma pigmentosum (XP) group A was studied and 3 nonsense mutations of the XP-A complementing gene (XPAC) were identified. One was a nucleotide transition altering the Arg-228 codon (CGA) to a nonsense codon (TGA). This transition creates a new cleavage site for the restriction endonuclease HphI. Of 21 unrelated Japanese XP-A patients examined, 1 (XP39OS) was a homozygote for this mutation and 3 were compound heterozygotes for this mutation and for the splicing mutation of intron 3 reported previously which is the most common mutation in Japanese patients and creates a new cleavage site for the restriction endonuclease AlwNI. The second mutation was a nucleotide transition altering the Arg-207 codon (CGA) to a nonsense codon (TGA). A Palestinian patient (XP12RO) who had severe symptoms of XP was homozygous for this mutation. The third mutation was a nucleotide transversion altering the Tyr-116 codon (TAT) to a nonsense codon (TAA). This transversion creates a new cleavage site for the restriction endonuclease MseI. Of the Japanese patients, 2 with severe clinical symptoms had this mutant allele. One was a compound heterozygote for this mutation and for the splicing mutation, and the other was heterozygous for this mutation and homozygous for the splicing mutation. Although most XP-A patients such as XP12RO have severe skin symptoms and neurological abnormalities of the de Sanctis-Cacchione syndrome, patient XP39OS was an atypical XP-A patient who had mild skin symptoms and minimal neurological abnormalities. Our results suggest that the clinical heterogeneity in XP-A is due to different mutations in the XPAC gene. Moreover, our data indicate that almost all Japanese cases of XP-A are caused by one or more of the 3 mutations, i.e., the splicing mutation of intron 3 and the 2 nonsense mutations of codons 116 and 228. Therefore, by restriction fragment length polymorphism analysis of PCR-amplified DNA sequences using the 3 restriction enzymes described above, rapid and reliable diagnosis of XP-A can be achieved in almost all Japanese subjects including prenatal cases and carriers.     

Ornithine transcarbamylase deficiency: new sites with increased probability of mutation

Ornithine transcarbamylase (OTC) deficiency, the most common inborn error of the urea cycle, shows an X-linked inheritance with frequent new mutations. Investigations of patients with OTC deficiency have indicated an overproportionate share of mutations at CpG dinucleotides. These statistics may, however, be biased because of the easy detection of CpG mutations by screening for TaqI and MspI restriction sites. In the present study, we investigated 30 patients, with diagnosed OTC deficiency, for new sites with an increased probability of mutation by complete DNA sequence analysis of all ten exons of the OTC gene. In six patients, two codons in exons 2 and 5, respectively, contained novel recurrent mutations, all of them affecting CpG dinucleotides. They included C to T and G to A transitions in codon 40, changing an arginine to cysteine and histidine, respectively, and a C to T transition in codon 178 causing the substitution of threonine by methionine. The first two mutations were characterized by a mild clinical course with high risk of sudden death in late childhood or early adulthood, whereas the third mutation showed a more severe phenotypic expression. In addition to these novel mutations, we identified four patients with the known R277W mutation, making it the most common point mutation of the OTC gene.     

Analysis of mutations in the p16/CDKN2A gene in sporadic and familial melanoma in the Polish population

Mutations in CDKN2A have been found in sporadic cutaneous malignant (CMM), in familial CMM and in other syndromes associated with melanoma. In this study DNA was obtained from 207 individuals and five cell lines. There were 157 CMM patients and 50 healthy members of melanoma patients families. The CMM group included patients with one or two melanoma cases in the family, families with dysplastic nevus syndrom (DNS) and patients with a spectrum of other types of cancers in the family. PCR-SSCP analysis and sequencing identified: six substitutions in codon 58 CGA/TGA (Arg/Stop), 16 substitutions GAC/GAT in codon 84 (Asp/Asp), six substitutions CGA/TGA in codon 148 (Arg/Thr), 14 substitutions G/C in 3'UTR and 4 double changes (two in codon 84 and 3'UTR; two in codon 148 and 3'UTR). The mutation identified in codon 58 was found in tissue only. Other substitutions were polymorphisms found in DNA from tissue and blood samples. Most of them were identified in sporadic CMM (six in codon 148 Ala/Thr, 12 in codon 84 Asp/Asp and six in 3'UTR). The frequency of the polymorphisms was also high in DNS and CMM/DNS families (four in codon 84 Asp/Asp and six in 3'UTR). No mutations or polymorphisms were found in CMM patients with one or two melanoma cases and CMM patients, with other cancers in family history. The analysis of the CDKN2A gene mutations in the Polish population demonstrated: (i) no germline mutations; (ii) a relatively high number of genetic changes in sporadic melanoma; (iii) a high number of polymorphisms in DNS and CMM/DNS families.     

Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene

Mutations at the factor VIII gene locus causing Haemophilia A have now been identified in many patients from many ethnic groups. Earlier studies used biased methods which detected repetitive mutations at a few CG dinucleotides. More recently rapid gene scanning methods have uncovered an extreme diversity of mutations. Over 80 different point mutations, 6 insertions, 7 small deletions, and 60 large deletions have been characterised. Repetitive mutation has been proved for at least 16 CpG sites. All nonsense mutations cause severe disease. Most missense mutations appear to cause instability of the protein, but some are associated with production of dysfunctional factor VIII molecules, thereby localising functionally critical regions of the cofactor. Variable phenotype has been observed in association with three of the latter class of genotype. This catalogue of gene lesions in Haemophilia A will be updated annually.     

Factor IXPortland: a nonsense mutation (CGA to TGA) resulting in hemophilia B

Male siblings with severe hemophilia b were studied for the molecular defect responsible for their disorder. To define the precise DNA alteration, a 362-bp fragment in the first part of exon VIII of the factor IX gene was amplified and sequenced. A single-base-pair substitution of C----T at the nucleotide sequence 30875 was found which resulted in a nonsense mutation (TGA) and terminated the protein synthesis of factor IX at amino acid residue 252. The single-base change occurred as a classic CG dinucleotide alteration to TG (or CA), a common mechanism for point mutations in mammals.     

Identification of factor IX mutations in haemophilia B: application of polymerase chain reaction and single strand conformation analysis

Summary The molecular characterization of two haemophilia B defects, Calgary 1 and Calgary 2, was carried out using polymerase chain reaction (PCR) amplification and direct dideoxy sequencing. It had been previously shown that the Calgary 1 mutation affects the 5 TaqI restriction site of exon VIII, whereas Calgary 2 involves the loss of the 3 TaqI site of exon VIII of the factor IX gene. Sequencing data has now revealed that each of these alterations involves a C-to-T transition within a CpG dinucleotide. In each instance an arginine residue is replaced by a stop codon. These cases represent the recurrence of each particular alteration, both of which are predicted to result in the production of a truncated protein lacking a significant part of the catalytic region. A recently developed technique that reveals base substitutions as single-strand conformation polymorphisms (SSCP) was adapted for modelling in the detection of point mutations. Referred to here as single-strand conformation (SSC) analysis, this procedure, used in association with PCR, provided a reliable and sensitive system for molecular diagnosis in each of the cases presented. Computer-generated secondary structure predictions demonstrated a strong correlation with experimental results and the technique was used to screen 11 additional patients in the same region. A change detected by SSC analysis in one patient was localized to 55 base pairs, sequenced, and identified as a conservative amino acid substitution. This patient is now referred to as Calgary 3.     

Replacement of arginine 773 by cysteine or histidine in the human androgen receptor causes complete androgen insensitivity with different receptor phenotypes

We have discovered two different point mutations in a single codon of the X-linked androgen-receptor (AR) gene in two pairs of unrelated families who have complete androgen insensitivity (resistance) associated with different AR phenotypes in their genital skin fibroblasts. One mutation is a C-to-T transition at a CpG sequence near the 5' terminus of exon 6; it changes the sense of codon 773 from arginine to cysteine, ablates specific androgen-binding activity at 37 degrees C, and eliminates a unique KpnI site at the intron-exon boundary. The other mutation is a G-to-A transition that changes amino acid 773 to histidine and eliminates an SphI site. This mutant AR has a normal androgen-binding capacity at 37 degrees C but has a reduced affinity for androgens and is thermolabile in their presence. Transient transfection of COS cells with cDNA expression vectors yielded little androgen-binding activity at 37 degrees C from Arg773Cys and abundant activity with abnormal properties from Arg773His, thereby providing the pathogenicity of both sequence alterations. This conclusion coincides with the following facts about evolutionary preservation of the position homologous to Arg773 in the AR: it is occupied by Arg or lysine in the progesterone, glucocorticoid, and mineralocorticoid receptors, and it is within a 14-amino-acid region of their steroid-binding domains that share approximately 85% amino acid identity.     

Identification of NF2 germ-line mutations and comparison with neurofibromatosis 2 phenotypes

Neurofibromatosis 2 (NF2) is an autosomal inherited disorder that predisposes carriers to nervous system tumors. To examine genotype-phenotype correlations in NF2, we performed mutation analyses and gadolinium-enhanced magnetic resonance imaging of the head and full spine in 59 unrelated NF2 patients. In patients with vestibular schwannomas (VSs) or identified NF2 mutations, the mild phenotype was defined as <2 other intracranial tumors and ≤ 4 spinal tumors, and the severe phenotype as either ≥ 2 other intracranial tumors or > 4 spinal tumors. Nineteen mutations were found in 20 (34%) of the patients and were distributed in 12 of the 17 exons of the NF2 gene, including intron-exon boundaries. Seven mutations were frameshift, six were nonsense, four were splice site, two were missense, and one was a 3-bp in frame deletion. The nonsense mutations included one codon 57 and two codon 262 C→T transitions in CpG dinucleotides. The frameshift and nonsense NF2 mutations occurred primarily in patients with severe phenotypes. The two missense mutations occurred in patients with mild phenotypes, and three of the four splice site mutations occurred in families with both mild and severe phenotypes. Truncating NF2 mutations are usually associated with severe phenotypes, but the association of some mutations with mild and severe phenotypes indicates that NF2 expression is influenced by stochastic, epigenetic, or environmental factors. Received: 4 July 1996     

Premature translation termination mediates triosephosphate isomerase mRNA degradation.

We characterized an anemia-inducing mutation in the human gene for triosephosphate isomerase (TPI) that resulted in the production of prematurely terminated protein and mRNA with a reduced cytoplasmic half-life. The mutation converted a CGA arginine codon to a TGA nonsense codon and generated a protein of 188 amino acids, instead of the usual 248 amino acids. To determine how mRNA primary structure and translation influence mRNA stability, in vitro-mutagenized TPI alleles were introduced into cultured L cells and analyzed for their effect on TPI RNA metabolism. Results indicated that mRNA stability is decreased by all nonsense and frameshift mutations. To determine the relative contribution of the changes in mRNA structure and translation to the altered half-life, the effects of individual mutations were compared with the effects of second-site reversions that restored translation termination to normal. All mutations that resulted in premature translation termination reduced the mRNA half-life solely or mainly by altering the length of the mRNA that was translated. The only mutation that altered translation termination and that reduced the mRNA half-life mainly by affecting the mRNA structure was an insertion that shifted termination to a position downstream of the normal stop codon.