Haemophilia B: database of point mutations and short additions and deletions, fifth edition, 1994.

The fifth edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of < 30bp) identified in haemophilia B patients. The 1,142 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.     

Haemophilia B: database of point mutations and short additions and deletions, 7th edition.

The seventh edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1535 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, presence of inhibitor and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.     

Haemophilia B: database of point mutations and short additions and deletions--eighth edition.

The eighth edition of the haemophilia B database (http://www.umds.ac. uk/molgen/haemBdatabase.htm ) lists in an easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1713 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, presence of inhibitor and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.     

Molecular pathology of haemophilia B.

Direct sequencing of amplified genomic DNA has been used to investigate the molecular basis of haemophilia B and thus identify specific amino acids that are essential for maintenance of structure or function of factor IX. Substitution of Cys 336, Asn 120 results in loss of circulating factor IX antigen and deletion of Arg 37 in gross reduction of circulating protein and loss of activity, while substitution of Arg -4, Arg 333, Asp 64 and Pro 55 cause loss of function without marked reduction in protein serum levels. Frameshift or point mutations resulting in marked loss of coding information are found in patients who develop antibodies to administered factor IX. An enhanced rate of mutation is evident at two CpG dinucleotides in the factor IX gene, which accounts for approximately 25% of all point mutations causing haemophilia B known to date. Direct sequencing of mutations also permits, for the first time, rapid and unequivocal prenatal and carrier diagnoses, in all cases, by eliminating the need for informative segregation of markers.     

Genetik und Klinik der Hämophilie A und B

Haemophilia A and B are caused by various mutations in the factor VIII (FVIII) and factor IX (FIX) genes, respectively. The clinical course of the disease is variable, dependent on the severity of the molecular defect. Nowadays, haemophilia patients can excellently be treated by plasma-derived or recombinant clotting factor concentrates. Thus, bleeding and its consequences can be almost completely prevented with nearly normal quality of life and life expectancy. The most severe complication of this treatment is the formation of antibodies (inhibitors) against the substituted clotting factor. The risk of inhibitor formation correlates significantly with specific mutation types that preclude endogenous factor VIII/IX protein synthesis and can be as high as 20–50%. The information on the expected clinical course is at present the most important indication for FVIII/IX gene analysis. Knowledge of the underlying FVIII/IX gene mutation further allows a reliable and fast carrier diagnosis in female relatives of patients with haemophilia.     

Determinants of the factor IX mutational spectrum in haemophilia B: an analysis of missense mutations using a multi-domain molecular model of the activated protein

A multi-domain molecular model of factor IXa was constructed by comparative methods. The quaternary structure of the protein was assembled by docking individual domains through consideration of their shape complementarity, polaric properties and the location of cross-reacting material positive/negative (CRM^+/–) variants on domain surfaces. Some 217 different missense mutations in the factor IX (F9) gene were then selected for study. Using maximum likelihood analysis, missense mutations affecting highly conserved amino acid residues of factor IX were shown to be 15–20 times more likely to result in haemophilia B than those affecting non-conserved residues. However, about one quarter of this increase in likelihood of clinical observation could be attributed to the magnitude of the amino acid exchange. Missense mutations in structurally conserved residues were found to be 2.1-fold more likely to come to clinical attention than those in structurally variable residues. Missense mutations in residues whose side chains were inwardly pointing were 3.6-fold more likely to be observed than those in surface residues. These observations imply a complex hierarchy of sequence/structure conservation in the protein. The severity of the clinical phenotype correlated with both the extent of the evolutionary sequence conservation of the residue at the site of mutation and the magnitude of the amino acid exchange. Further, the substitution of residues exhibiting minimal side chain solvent accessibility was associated disproportionately with severe haemophilia compared with that of surface residues. Clusters of CRM⁺ mutations were observed at factor IX-specific residues on the surface of the molecule. These clusters may reflect factor IX-specific docking interactions. The likelihood that a given factor IX mutation will come to clinical attention is therefore a complex function of the sequence characteristics of the F9 gene, the nature of the amino acid substitution, its precise location and immediate environment within the protein molecule, and its resulting effects on the structure and function of the protein.This paper is dedicated to the memory of Andrew Wacey     

Recurrent mutations in the factor IX gene: founder effect or repeat de novo events

The investigation of 114 unrelated patients, representing about half the sample of the German haemophilia B population, enabled us to delineate the causative mutation in 103 (90.4%) haemophilic factor IX genes. Of these 103 cases 84 (81.6%) turned out to be unique molecular events, the remainder being repeats. Haplotype analysis revealed that the great majority, if not all, of these recurrent observations occurred independently. This conclusion is supported by our finding that three de novo mutations could be demonstrated at two sites of frequent mutation. A further 20 de novo events could be established in an unselected sample of 37 families with sporadic haemophilia B and 37 families with a history of the disease. Altogether, the germ line of origin could be determined in 21 of these 23 cases, thereby indicating a ratio of male to female mutation rates close to 2. On the basis of the data available, it is becoming clear that rearrangements in the factor IX gene (35.4% of de novo cases) are responsible for haemophilia B at a higher frequency than has been observed today (12.3%). More than two-thirds of the de novo cases cause the severe form of the disease, thereby reflecting the deficit of these haemophilic genes in the actual gene pool because of excess mortality in the past. In addition 40% (12/30) of the de novo single-base mutations were transitions at CpG dinucleotides. Compared with the expected at-random frequency, this observation indicates an 83-fold enhancement of mutation at CpG.     

Direct detection of point mutations by mismatch analysis: application to haemophilia B.

Rapid detection of point mutations in genomic DNA has been achieved by chemical mismatch analysis of heteroduplexes formed between amplified wild-type and target sequences in the human factor IX gene. Amplification and mismatch detection (AMD) analysis of DNA from relatives of haemophilia B patients permitted carrier diagnosis by direct identification of the presence or absence of the mutation in all cases, thus eliminating the need for the informative segregation of polymorphic markers. This extends diagnostic capability to virtually all haemophilia B families. AMD analysis permits detection of all sequence variations in genomic DNA and is therefore applicable to direct diagnosis of X-linked and autosomal diseases and for identification of new polymorphisms for genetic mapping.     

Diagnosis of haemophilia B using the polymerase chain reaction

The polymerase chain reaction (PCR) was used to amplify specific DNA sequences within the factor IX gene of haemophilia B patients and their relatives. Three of the amplified fragments contain polymorphic sites, which can be used as markers in segregation analyses. These restriction fragment length polymorphisms (RFLPs) were until recently detected by Southern blotting after digestion with the restriction enzymes Taq I, Dde I and Xmn I. All three RFLP's are located in introns of the factor IX gene and together are informative in approximately 70% of all cases. Each of the polymorphisms was successfully used in carrier detection studies after amplification of the relevant fragments. This method is also suitable for rapid antenatal diagnosis. Additionally we were able to amplify all eight exons of the factor IX gene including the splice junctions and a part of the 5'-region. Large deletions or insertions can be detected without further analysis. Several possibilities for the rapid detection of point mutations after DNA amplification have been described recently. The complete amplification of all functional parts of the Factor IX gene in combination with these new techniques should enable us to detect the majority of mutations leading to haemophilia B.     

A factor IX mutation, verified by direct genomic sequencing, causes haemophilia B by a novel mechanism.

A novel factor IX gene mutation (factor IX London 2) has been characterized. This causes severe crm+ haemophilia B as the patient's plasma shows normal factor IX antigen level and less than 1% clotting activity. Sequence analysis of the entire cloned coding and promoter regions revealed a single point mutation: a G----A transition at position 31,119. This region of the patient's DNA was amplified in vitro by the polymerase chain reaction and the nucleotide change was confirmed by direct sequencing of the amplified products. The mutation results in the substitution of the arginine at position 333 by glutamine. This arginine residue is absolutely conserved in the catalytic domain of normal human and bovine factor IX, X and prothrombin. The substitution by glutamine causes the loss of a positive charge from the surface of the factor IX London 2 protein. This mutation pinpoints a previously unknown, functionally critical feature of factor IX which may be involved in substrate or co-factor binding.     

Missense mutations and evolutionary conservation of amino acids: evidence that many of the amino acids in factor IX function as "spacer" elements.

We report 31 point mutations in the factor IX gene and explore the relationship between the level of evolutionary conservation of an amino acid and the probability of a mutation causing hemophilia B. From our total sample of 125 hemophiliacs and from those reported by others, we identify 95 independent missense mutations, 94 of which occur at amino acids that are evolutionarily conserved in the available mammalian factor IX sequences. The likelihood of a missense mutation causing hemophilia B depends on whether the residue is also conserved in the factor IX-related proteases: factor VII, factor X, and protein C. Most of the possible missense mutations in generically conserved residues (i.e., those conserved in factor IX and in all the related proteases) should cause disease. In contrast, missense mutations in factor IX-specific residues (i.e., those conserved in human, cow, dog, and mouse factor IX but not in the related proteases) are sixfold less likely to cause disease. Missense mutations at nonconserved residues are 33-fold less likely to cause disease. At least three models are compatible with these observations. A comparison of sequence alignments from four and nine species of factor IX and an examination of the missense mutations occurring at CpG residues suggest a model in which most residues fall on opposite ends of a spectrum. In about 40% of residues, virtually any missense mutation in a minority of the residues will cause disease, while virtually no missense mutations will cause disease in most of the remaining residues. Thus, many of the residues in factor IX are spacers; that is, the main chains are presumably necessary to keep other amino acid interactions in register, but the nature of the side chain is unimportant.     

Human germline mutation in the factor IX gene

The molecular epidemiology of factor IX germline mutations in patients with hemophilia B has been studied in detail because it is an advantageous model for analyzing recent germline mutations in humans. It is estimated that mutations have been defined in the majority of nucleotides that are the target for mutation. The likelihood that a factor IX missense mutation will cause disease correlates with the degree of evolutionary conservation of the amino acid. Mutation rates per base-pair have been estimated after careful consideration and correction for biases, predicting about 76 de novo mutations per generation per individual resulting in 0.3 deleterious changes. The male-to-female sex ratio of mutation varies with the type of mutation. There is evidence for a maternal age effect and an excess of non-CpG G:C to A:T transitions. The factor IX mutation pattern is similar among geographically, racially and ethnically diverse human populations. The data support primarily endogenous mechanisms of germline mutation in the factor IX gene. Mutations at splice junctions are compatible with simple rules for predicting disease causing mutations.     

An Excess of G over C Nucleotides in Mutagenesis of Human Genetic Diseases

Strand asymmetries in DNA evolution, including indel and single nucleotide substitutions, were reported in prokaryotes. Recently, an excess of G>A over C>T substitutions in hemophilia B patients was recognized in our molecular diagnostic practices. Further analysis demonstrated biased point mutations between sense and antisense strands when unique changes in factor IX were counted. Similar mutation spectra of factor IX and the HGMD prompted us to speculate that the excess of G>A over C>T may be present in genes other than factor IX. Data from nine genes (each has ≥100 missense mutations) retrieved from HGMD, international factor IX database, and Dr. Sommer’s lab database in the City of Hope National Medical Center, Duarte, CA, USA were analyzed for their point mutation spectra. Similar to factor IX, all genes selected in this study have biased G>A over C>T unique mutations when nonsense mutations were excluded. The biased missense point mutations were recently convincingly documented by the statistic data of categorized missense mutation in HGMD. The consistence of the genetic observation and the genomic data from HGMD strongly indicate that biased point mutations, possibly a phenotypic selection, are more widespread than previously thought. The biased mutations have immediate clinical impact in molecular diagnostics.     

Treatment of haemophilia and related disorders in Britain and Northern Ireland during 1976-80: report on behalf of the directors of haemophilia centres in the United Kingdom.

A five year survey of the treatment of patients in the United Kingdom suffering from haemophilia and related disorders was carried out on behalf of the directors of haemophilia centres. The survey showed an increase in the number of patients receiving treatment from the centres, a substantial increase in the total amount of therapeutic materials used, and an increase in the average amount of factor VIII or factor IX used yearly per patient. Home treatment became established for severely affected patients and accounted for roughly half of the total amount of material used. Study of the acquisition of factor VIII or factor IX antibodies (inhibitors) in patients with haemophilia A or haemophilia B showed no increase in antibodies during the survey period, despite the increased use of factor VIII and factor IX concentrates. The occurrence of acute hepatitis in treated patients was also studied and no increased incidence was observed. A near normal median expectation of life in patients with severe haemophilia A was found.     

The molecular pathology of haemophilia B. Fourth Wellcome Trust lecture

Haemophilia is a rare inherited disease of blood clotting known since biblical times. The rarer form (haemophilia B) occurs in about 1 in 30,000 males and there are about 900 patients in the U.K. at present. Biochemically, patients either lack or have a defective protein (called factor IX) which is needed for the clotting of blood in response to injury. Only males get the disease. However, females can carry the trait in a latent form and transmit the disease to their offspring. Untreated, the disease leads to internal bleeding into muscles and joints and is life-threatening. In the U.K. and in countries with effective health care programmes, patients are treated by periodic injection of factor IX concentrate, a drug isolated from the pooled plasma derived from many blood donors. This drug replaces their own absent or defective factor IX and allow them to enjoy a relatively normal lifestyle. I have reviewed recent studies on the molecular genetics of haemophilia B which started with the isolation of the gene coding the factor IX protein from normal individuals in 1984. Following this, it has been possible firstly to produce factor IX artificially in the laboratory from cloned copies of the messenger RNA of the factor IX gene. Secondly, it has been possible to improve the diagnosis of 'carriers'. Carrier females often wish to know whether they are carriers or not before they have children. If they are positively identified as carriers, the risk and implications of having a haemophiliac son can be discussed and therapeutic abortion considered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular analysis of hemophilia B in Poland: 12 novel mutations of the factor IX gene

We examined the molecular basis of factor IX deficiency in 53 unrelated Polish patients with hemophilia B. Heteroduplex analysis and direct sequencing of polymerase chain reaction (PCR) products were applied to identify the gene defect. Forty-three different point mutations were detected in the factor IX gene of 47 patients. There were 29 missense mutations, 9 nonsense mutations, 4 splice site mutations and 1 point mutation in the promoter region. Twelve mutations were novel. The results of this study emphasize a very high degree of heterogeneity of hemophilia B.     

Coumarin-induced abnormal factor IX: an immunological study in humans.

An excess of factor IX antigen or protein with respect to factor IX activity is present in coumarin treated patients. The average factor IX antigen value found in a group of 16 patients was 96.2 (S.D. = 24.46) whereas the average clotting activity was 19 (S.D. = 4.54) (p less than 0.001). In the electroimmunoassay system a normal peak or precipitate were seen in every instance. In haemophilia B--no peak or precipitate were seen. The coumarin induced abnormal factor IX shows a more anodic migration in the bidimensional immunoelectrophoresis system as compared with the normal counterpart. On the contrary, the factor IX protein present in haemophilia BM or in haemophilie B+ migrates as normal factor IX.     

Protein engineering of the propeptide of human factor IX

Vitamin-K-dependent plasma proteins contain a highly conserved propeptide sequence located between the classical hydrophobic leader sequence and the N-terminus of the mature protein. This acts as a recognition sequence for the vitamin-K-dependent carboxylase which catalyses the conversion of specific glutamate residues to gamma-carboxyglutamate (Gla) residues in the adjacent Gla domain. Protein engineering of the 18 residue propeptide from human factor IX has highlighted the importance of residues -16Phe and -10Ala with respect to carboxylase recognition. In addition, studies of haemophilia B patients have shown that C-terminal propeptide residues -4Arg and -1Arg are required for proteolysis of the propeptide from the mature protein. To extend these previous studies we have introduced two novel mutations into the propeptide of human factor IX at positions -17(Val----Asp) and -6(Leu----AsP), and studied the effect of these changes on gamma-carboxylation and proteolytic processing. Both mutations reduce the expression of a calcium-dependent epitope in the Gla domain; however, only -6Leu----Asp shows reduced binding to barium sulphate. In addition, this latter mutation prevents proteolytic processing of the propeptide. These data support the current hypothesis that the propeptide contains two recognition elements: one for carboxylase recognition located towards the N-terminus, and one for propeptidase recognition located near the C-terminus.     

Assessing the underlying pattern of human germline mutations: lessons from the factor IX gene.

Germline mutations cause or predispose to most disease. Hemophilia B is a useful model for studying the underlying pattern of recent germline mutations in humans because the observed pattern of mutation in factor IX more closely reflects the underlying pattern of mutation than the observed pattern for many other genes. In addition, it is possible to identify and correct for biases inherent in ascertaining only those mutations that cause hemophilia. Aspects of the pattern of germline mutation in the factor IX gene are becoming clear: 1) in the United States, two-thirds of mutations causing mild disease arose from three founders whereas almost all the mutations resulting in either moderate or severe disease arose independently, generally within the past 150 years; 2) direct estimates of the rates of mutation in humans indicate that transitions are more frequent than transversions, which in turn are more frequent than deletions and insertions; 3) transitions at CpG are elevated approximately 24-fold relative to transitions at non-CpG dinucleotides; 4) transversions at CpG are elevated approximately eightfold relative to transversions at non-CpG dinucleotides; 5) the sum total of the dinucleotide mutation rates produces a bias against G and C bases that would be sufficient to maintain the G+C content of the factor IX gene at its evolutionarily conserved level of 40%; and 6) the pattern of mutation is similar for Caucasians residing in the United States and for Asians residing in Asia. Two ideas emerge from this and from an analysis of the pattern of recent deleterious mutations compared with ancient neutral mutations that have been fixed during evolution into the factor IX gene. First, the bulk of germline mutations are likely to arise from endogenous processes rather than environmental mutagens. Second, the factor IX protein is composed mostly of two classes of amino acids: critical residues in which all single-base missense changes will disrupt protein function, and "spacer" residues in which the precise nature of the residue is unimportant but the peptide bond is necessary to keep the critical residues in register. More work is necessary to assess the veracity and generality of these ideas.