cytidylyl-3--5--guanosine and Hemophilia-A

CpG dinucleotides are efficiently methylated in vertebrate genomes except in the CpG islands having a high C+G content. Methylated CpGs are the single most mutated dinucleotide. Sequences surrounding disease causing CpG mutation sites were analyzed from locus-specific mutation databases. Both tetra- and heptanucleotide analyses indicated clear overall sequence preference for having pyrimidines 5' and purines 3' to the mutated 5-methylcytosine. The most mutated tetranucleotides are TCGA and TCGG, the former being also a frequent restriction and modification site. The results will help in elucidating the still controversial mutation mechanism of CpG doublets.

Topics: Agammaglobulinaemia Tyrosine Kinase; Agammaglobulinemia; Base Sequence; Dinucleoside Phosphates; DNA Methylation; Factor VIII; Genes, p53; Genetic Linkage; Hemophilia A; Molecular Sequence Data; Mutation; Oligodeoxyribonucleotides; Phenylalanine Hydroxylase; Phenylketonurias; Point Mutation; Protein-Tyrosine Kinases; X Chromosome

The mutations of 76 haemophilia B patients representing the whole population registered with the Malmö haemophilia centre (42) and referrals from the UK, were characterised. RFLP haplotype analysis of the defective genes indicated that 51 single base pair substitutions were definitely of independent origin and 27 of these were CpG----TpG or CpA transitions. This represents a 38-fold excess over other single-base changes. Most of such transitions (82%) occur at 9 CpG sites occupying critical positions (transitions at 3 sites substitute essential arginines, while at 6 sites transition to TpG creates stop codons). Sixteen of the 18 possible transitions at these 9 sites cause clear haemophilia B and should be fully ascertained in our haemophilia B population. This allowed the direct estimate of the rate of CpG transitions. This is 1.05 x 10(-7) substitutions per base per gamete per generation. The marked excess of CpG transitions in haemophilia B appears partly due to the high proportion of CpG sites at critical positions (at least 9 out of 20). We propose that this follows from the fact that male hemizygosity makes X-linked genes particularly susceptible to selective forces that tend to fix CpG sites arising at critical positions.

Topics: Amino Acids; Dinucleoside Phosphates; Exons; Factor IX; Genes; Haplotypes; Hemophilia A; Humans; Mutation; Polymorphism, Restriction Fragment Length

We have used the polymerase chain reaction (PCR) and differential oligonucleotide melting to screen for mutations in selected CpG dinucleotides in the factor VIII genes of haemophilia A patients. By this means we have identified and confirmed by sequencing a novel point mutation in codon 372 (CGC) of the factor VIII gene of a moderately severe CRM+ haemophiliac. The first C of this codon has been substituted by T resulting in the non-conservative substitution of cysteine for arginine at an essential thrombin cleavage site in factor VIII. Analysis of three intragenic restriction fragment length polymorphisms was uninformative in the patient's family. However, DNA analysis for the specific mutation shows one sister and the patient's mother to be carriers, and the other sister to be normal. This DNA analysis confirmed the results of phenotype analysis by factor VIII coagulant to von Willebrand factor antigen ratios for the females at risk. The two carrier females had low factor VIII coagulant activity and excess VIII antigen as predicted but the non-carrier sister also had anomalously high VIII antigen in her plasma. When feasible, mutation specific DNA analysis is able to resolve the difficulties posed by variable phenotype data and unknown level of mutation in sporadic haemophilia A.

Topics: Base Sequence; Dinucleoside Phosphates; DNA Mutational Analysis; Factor VIII; Family; Female; Genetic Carrier Screening; Hemophilia A; Humans; Male; Molecular Sequence Data; Mutation; Pedigree; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Thrombin

We have identified a CpG island contained within the largest factor VIII intron. This island is associated with a 1.8-kb transcript and, unlike factor VIII, is produced abundantly in a wide variety of cell types. The nested gene is oriented in a direction opposite to that of factor VIII and contains no intervening sequences. A cDNA of 1739 bases was isolated from a human liver library and found to have a GC-rich, long open reading frame. Two computer-assisted methods (Fickett TESTCODE and Staden-McLachlan codon usage) predict that the gene codes for a protein. Two other copies of this gene are located within 1.1 Mb of the factor VIII gene. Northern blot analysis of RNA isolated from hemophilia patients deleted for factor VIII sequences has shown that both the intron gene and at least one other copy of the gene are transcribed. A homologous, transcribed sequence is also present in mice.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Blotting, Southern; Cell Line; Dinucleoside Phosphates; Factor VIII; Genes; Hemophilia A; Humans; Introns; Molecular Sequence Data; Restriction Mapping; Transcription, Genetic