Haemophilia B: database of point mutations and short additions and deletions--second edition.
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Base Sequence↗
Biomedical subjects
F Giannelli
Publications and source records attributed to F Giannelli.
Detection of three novel mutations in two haemophilia A patients by rapid screening of whole essential region of factor VIII gene.
In an attempt to replace the existing, DNA-based, 50% effective, carrier and prenatal diagnoses of haemophilia A with the 100% successful direct detection of defective genes, a new procedure was developed to screen and identify mutations in all the essential regions of the factor VIII gene (putative promoter, coding sequence, and the cleavage and polyadenylation region). Genomic DNA and cDNA obtained by reverse transcription of the "leaky" mRNA found in peripheral lymphocytes were amplified by means of the polymerase chain reaction to yield a set of eight segments comprising the essential gene sequences. The segments were then screened individually for mutations by the amplification mismatch detection method, which detects and locates any type of sequence discrepancy between the test DNA and the control probe by cleavage of the probe at the site of mismatches. Two haemophilia A patients were studied. The first showed two single-base changes: one (substitution of tryptophan 2229 by cysteine in the C2 domain) is the probable cause of the disease, since it affects a conserved residue of factor VIIIa, whereas the other (the conservative substitution of aspartic acid at position 1241 by glutamic acid) occurs in a domain (B) irrelevant to factor VIII activity. The second patient showed a complete failure of pre-mRNA splicing due to a single-base substitution that changes the obligatory AG acceptor splice site of intron 5 to GG. The method characterises the gene defect in 10 days or less and should lead to the rapid accumulation of information on the molecular biology of haemophilia A.
Adult↗
Cooperation between human cells sensitive to UVA radiations: a clue to the mechanism of cellular hypersensitivity associated with different clinical conditions.
Six fibroblast strains sensitive to long wavelength ultraviolet radiation (UVA) and one control strain were used to see if cooperation between the different cell strains could modify the abnormally high yield of single-strand DNA breaks (SSB) in the sensitive strains caused by UVA irradiation in complete Dulbecco's MEM. The sensitive strains were established from individuals showing proneness to different types of light-induced skin damage (actinic reticuloid, familial actinic keratoses with internal malignancies, and unusual frequency of basal cell carcinomata). When sensitive and normal cells were cocultivated, the UVA-induced SSB decreased in the sensitive cells and increased in the normal ones by amounts proportional to the ratio of the two types of cells in the mixtures. Furthermore the regression of SSB, in the sensitive cells, on the proportion of normal cells in the mixture extrapolated to normal levels of SSB when the proportion of normal cells increased to one. Cocultivation of different sensitive cells did not reduce the UVA-induced SSB to levels below those of the less sensitive cell strains. From these results we conclude that substances, present in limiting amounts, even in normal cells, can be transferred from cell to cell, presumably by metabolic cooperation, and modify the yield of SSB caused by UVA radiation. The abnormal yields of SSB in the sensitive cells appear to be entirely attributable to deficits in the substances responsible for the intercellular cooperation. We suggest that such substances are small molecular weight scavengers of active oxygen species.
Basal Cell Carcinoma↗
Genetics and molecular biology of haemophilias A and B.
The development of rapid procedures for the characterization of mutations is advancing the knowledge of the molecular biology of the haemophilias and transforming the strategies for the diagnoses required for genetic counselling. In haemophilia B more than 300 mutants have been fully characterized. These comprise complete and partial deletions, rare insertions, and 'point' mutations. The latter may impair transcription (promoter mutations), RNA processing (splicing mutations) and translation (frameshifts and stop codons) or cause single amino acid (aa) changes. Eighty-four residues are involved in the 105 presumed detrimental aa substitutions reported so far and these are usually conserved in the factor IX homologues (factors VII, X and protein C) and/or the factor IX of different mammalian species. There are clear correlations between the mutation and clinical features. In addition mutations causing gross physical or functional loss of coding information appear to predispose to the development of antibodies against therapeutic factor IX. Hotspots of mutations have been identified and are usually associated with CpG sequences. In haemophilia A the size and complexity of the factor VIII gene has hindered the analysis of mutants. Most of the studies published so far have analysed only a small fraction of the essential region of the factor VIII gene and this led to the repeated observation of specific types of mutation. The recent development of a rapid method to analyse RNA splicing and the whole coding region of the factor VIII gene should unblock this situation. With regard to genetic counselling, the direct detection of gene defects has increased the proportion of haemophilia B families that can be helped from 60% to virtually 100% and similar expectations may now be formulated for haemophilia A. In the UK a national database of haemophilia B mutations is being constructed to optimize genetic counselling. This should offer a model for a similar development in haemophilia A.
DNA Mutational Analysis↗
Haemophilia B mutations in a complete Swedish population sample: a test of new strategy for the genetic counselling of diseases with high mutational heterogeneity.
Carrier and prenatal diagnosis based on the identification of the gene defect (direct diagnosis) increases the proportion of haemophilia B families that can be offered precise genetic counselling from the 50-60% attainable by DNA markers, to 100% and they also provide information on the molecular biology of the disease. We propose that in order to maximize the practical and scientific benefits of direct diagnosis the gene defect of complete (possibly national) populations of patients should be characterized and the information stored in appropriate confidential databases. We demonstrate the feasibility of such a strategy by characterizing the mutations of all the patients registered with the Malmö haemophilia centre. These patients (44 male and 1 female) are from 45 unrelated families and 24 (53%) have negative family history. The 25 patients with similar reduction of factor IX:C and factor IX:Ag (24 male + 1 female) have: two gross deletions, three frameshifts, four translation stops, six mutations expected to affect pre-mRNA splicing and 10 amino acid substitutions. The six patients with greater reduction of factor IX:C than factor IX:Ag and the seven with reduced IX:C and normal IX:Ag have only amino acid substitutions. Patients with inhibitors have: one complete deletion, one frameshift and three translation stops. One patient has both a translation stop and a functionally neutral amino acid substitution (His257----Tyr). Characterization of the factor IX mutation was successful in every case, usually entailed 4 person-days work, and has led to the identification of 12 amino acid residues essential for the factor IX structure and function.
Base Sequence↗
Moderate haemophilia B in a female carrier caused by preferential inactivation of the paternal X chromosome.
The case of a female with moderate haemophilia B is reported. She is the only affected member of her family, and factor IX RFLP analysis shows her to have inherited no maternal markers for polymorphisms located in the first intron and 8 Kb 3' of the polyadenylation signal (DdeI and HhaI, respectively). This clearly indicates a deletion involving at least the last 7 exons of the factor IX gene. Her other factor IX gene inherited from her healthy father is normal as her son is also healthy. This suggests the patient's haemophilia to be due to gross bias in the proportion of factor IX-producing cells with an inactive paternal X chromosome. Methylation studies on the 5' region of the PGK gene show that virtually all the patient's lymphocytes carry a hypermethylated and presumably an inactive paternal X chromosome. The reason for this bias in the activity of her two X chromosomes is not clear, as no chromosomal alterations were found.
Adult↗
A new strategy for carrier and prenatal diagnosis and molecular studies in haemophilia B.
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Chromosome Deletion↗
HIV-1 infection, tuberculosis, and syphilis in male transsexual prostitutes in Milan, Italy.
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Adult↗
Haemophilia B: database of point mutations and short additions and deletions.
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Base Sequence↗
The incidence and distribution of CpG----TpG transitions in the coagulation factor IX gene. A fresh look at CpG mutational hotspots.
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.
Amino Acids↗
Two factor IX mutations in the family of an isolated haemophilia B patient: direct carrier diagnosis by amplification mismatch detection (AMD).
Rapid identification of gene defects allows definite carrier and prenatal diagnosis in virtually every family with haemophilia B. We report a study of the family of an isolated patient. Analysis of all the essential regions of the patient's factor IX gene (promoter, exons, transcript processing signals) revealed two mutations: one C----T transition at residue 17762 and another at residue 30890. The former created a translation stop at codon 116, and the latter caused substitution of His 257 by Tyr. The translation stop is an obvious detrimental mutation, while the His 257----Tyr substitution has uncertain functional consequences. From analysis of other family members, it was found that the first mutation had occurred at grandpaternal gametogenesis, in keeping with the negative family history, while the second was of more remote origin and did not reduce the maternal grandfather's factor IX coagulant and antigen level. This neutral mutation (His 257----Tyr) pinpoints a poorly conserved amino acid in factor IX and related serine proteases. Its coexistence with a detrimental mutation stresses the need to examine all essential regions of a gene before attempting to interpret the functional consequences of its sequence changes.
Antigens↗
HIV-related severe thrombocytopenia in intravenous drug users: prevalence, response to therapy in a medium-term follow-up, and pathogenetic evaluation.
Severe thrombocytopenia (TP) accounted for 5.3% of cases in a consecutive series of 380 HIV-infected intravenous drug users (IVDUs) at presentation. Forty-one of 53 subjects with severe TP showed haemorrhages and were treated as follows: ten were splenectomized, 17 were given high-dose intravenous immunoglobulins (HDIg), and 10 received anti-Rh(D) immunoglobulins (anti-Rh Ig). Splenectomy induced a complete clinical response in all cases: four out of 10 patients maintained platelet counts greater than 100 x 10(9)/l. HDlg gave a good clinical response in all patients, but eight out of 17 suffered haemorrhages during the follow-up and recall treatments were necessary. Six out of 10 patients treated with anti-Rh lg maintained platelet counts greater than 30 x 10(9)/l, but in two cases the treatment was interrupted because of severe haemolysis. No patient progressed to overt AIDS during the follow-up. Splenectomized patients in particular did not show adjunctive risks of worsening of the HIV-related clinical picture. A platelet kinetic study performed in 20 patients with severe HIV-related TP suggests a possible role for platelet sequestration in TP of HIV-infected IVDUs, in which a liver involvement is very frequent.
Adult↗
The use of DNA amplification for genetic counselling related diagnosis in haemophilia B.
A single base pair variation in the coding sequence of coagulation factor IX produces a protein polymorphism detectable with monoclonal antibodies and a restriction fragment length polymorphism (RFLP). This allows carrier and prenatal diagnoses in 48% of Caucasian families segregating for haemophilia B. However, this RFLP cannot be detected by standard Southern blotting, while the antibody assay may give equivocal results in some females and can only allow prenatal diagnoses on second trimester fetal blood samples. We show that, using the polymerase chain reaction, the polymorphic DNA segment can be amplified and directly tested for the presence of the alternative sequences by a non-radioactive procedure that has the advantage of speed (1-2 days), partial automation and applicability to first trimester diagnoses. We also show that the method gives results on a single drop of dried blood.
Base Sequence↗
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.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
Familial keratoses of actinic distribution associated with internal malignancy and cellular hypersensitivity to UVA.
In members of a family there appeared to be an association between the development of cutaneous pigmented keratoses in sun-exposed sites, and the later evolution of internal malignancies, particularly carcinoma of the uterus. Affected individuals were not clinically photosensitive, but their fibroblasts demonstrated gross cytopathic changes, low survival indices and an increased frequency of DNA single strand breaks following exposure to long-wave ultraviolet radiation (UVA). These clinical and cellular features appear to identify an unrecognized syndrome that may not be uncommon. Recognition of this syndrome in a family may prevent certain malignancies developing in those affected.
Adult↗
Early onset Cockayne's syndrome: case reports with neuropathological and fibroblast studies.
Two patients with early onset Cockayne's syndrome are presented. In each case there was a striking failure of growth and developmental deterioration around six months of age. It has been suggested that early onset Cockayne's syndrome is a syndrome distinct from Cockayne's syndrome, but when the first patient died aged two years 10 months, examination of the brain showed a leucodystrohy with 'tigroid' demyelination similar to that reported in later onset cases of Cockayne's syndrome. Studies of the effects of UV irradiation on cultured fibroblasts from patients showed similar levels of inhibition of RNA synthesis to those seen in a control with Cockayne's syndrome. This evidence suggests it is appropriate to classify early onset Cockayne's syndrome with Cockayne's syndrome. Since there is a phenotypic overlap between early onset Cockayne's syndrome and COFS syndrome, they may both be classified within the same diagnostic group, but as yet no cellular studies with UV irradiation have been performed in COFS syndrome.
Brain↗