Haemophilia A mutations in the UK: results of screening one-third of the population.

One-third of the UK haemophilia A population was screened to establish a national database of mutations and pedigrees and advance knowledge of the disease. The following mutations were found: 131 intron 22- and 13 intron1-breaking inversions; 11 gross deletions and an insertion; 65 frameshifts; three in-frame deletions and one insertion; 46 nonsense; 30 intronic mutations affecting splice sites and four generating new sites; 469 non-synonymous mutations due to 203 different base substitutions of which four affected, and nine were predicted to affect, splicing; three promoter mutations; two synonymous exon 14 mutations possibly affecting splicing; two VWF mutations. Of the above mutations, 176 are not listed in the Haemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS). Four gross deletions arose by non-homologous end-joining; we detected unexpected splicing in some mutations; substitution of amino acids conserved for less than 90 million years are rare; the risk of developing inhibitors for patients with nonsense mutations is greater when the stop codon is in the 3' half of the mRNA; changes likely to generate splice sites causing frameshifts are over-represented among non-synonymous mutations associated with inhibitors; our data and those in HAMSTeRS enabled the size of the spectrum of specific mutations causing the disease to be estimated and to determine how much of it is known.

Int22h-related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test for their detection.

BACKGROUND: Intrachromosomal, homologous recombination of the duplicon int22h-1 with int22h-2 or int22h-3 causes inversions accounting for 45% of severe hemophilia A, hence the belief that int22h-2 and int22h-3 are in opposite orientation to int22h-1. However, inversions involving int22h-2 are five times rarer than those involving its virtually identical copy: int22h-3. Recent sequencing has indicated that int22h-2 and int22h-3 form the internal part of the arms of an imperfect palindrome so that int22h-2, in the centromeric arm, has the same orientation as int22h-1 and, upon recombination with int22h-1, should produce deletions and duplications but not inversions. AIM: This work aims to provide rapid tests for all the mutations that can result from recombinations between the int22h sequences and to investigate whether int22h-2-related inversions causing hemophilia A arise in chromosomes, where the arms of the palindrome have recombined so that int22h-2 and int22h-3 swap places and orientation. PATIENTS/METHODS: Twenty patients with int22h-related inversions were examined together with a control and inversion carriers using reverse transcription-polymerase chain reaction (RT-PCR), long-range PCR and sequencing. RESULTS AND CONCLUSIONS: Analysis of mRNA in patients and a control provided evidence confirming the palindromic arrangement of int22h-2 and int22h-3 and the proposed inversion polymorphism that allows int22h-2 to be in the telomeric arm of the palindrome and in opposite orientation to int22h-1. New long-range PCR reactions were used to develop a single tube test that detects and discriminates inversions involving int22h-2 or int22h-3 and a two-tube test that can distinguish inversions, deletions, and duplications due to recombination between int22h sequences.

Creation of a novel donor splice site in intron 1 of the factor VIII gene leads to activation of a 191 bp cryptic exon in two haemophilia A patients.

We have constructed a confidential U.K. database of haemophilia A mutations and pedigrees by characterizing the gene defect of one index patient in each U.K. family. Mutations were identified by screening all coding regions of the factor VIII (FVIII) mRNA, using solid-phase fluorescent chemical cleavage of mismatch and examining additional non-coding regions of the gene. Here we report two haemophilia A patients (UK 114 FVIII:C 2% and UK 243 FVIII:C < 1%) with an abnormal FVIII mRNA due to an A to G point mutation, 1.4 kb downstream from exon 1 in the FVIII gene. This mutation creates a new donor splice site in intron 1 and leads to insertion of a 191 bp novel exon in the mRNA. Haplotype analysis suggests that the mutation may have originated in a common ancestor of the two patients, who further illustrate how mRNA analysis allows higher efficiency of haemophilia A mutation detection, because their mutation would not have been identified by direct analysis of the factor VIII gene.

Mutation analysis and genetic service: the construction and use of national confidential databases of mutations and pedigrees.

The development of rapid mutation screening procedures allows the detection of mutations in large populations. This is particularly useful for inherited diseases of high mutational heterogeneity, such as haemophilia A and B, because the analysis of the very many different natural mutants clearly defines the features that are important to the function of the relevant gene and gene product. Furthermore, the characterization of the mutation in an index person from each affected family may lead to the construction of confidential databases of mutations and pedigrees that allow optimization of genetic service. We report how, motivated by the aforementioned concepts, we have planned and introduced in the UK a national strategy to optimize genetic service in both haemophilias and, in particular, we describe the principles that have guided us.

A problem with modern dental materials.

It may come as a surprise to learn that there is more than one discipline in medicine dealing with artificial organs. The obvious one is bio-engineering, which is largely concerned with fashioning existing biologically acceptable materials into useful spare parts such as hip or knee joints. The materials themselves hardly change, and the difference between rival prostheses lies more in design than novel chemistry. This is the world of titanium or cobalt-chromium joint designs, bone screws and plates, orthotic limbs, supports and wheelchairs, and futuristic ideas such as miniature video cameras for artificial eyes.

Cranio-maxillary fixation using supra-orbital pins.

This paper compares the forces which external frames, used for cranio-maxillary fixation, can resist. Clinically, several frames using supra-orbital pins have been proven satisfactory. By quantifying the resistance forces, a more objective assessment of their effectiveness is made. The resistance to backward displacement of the maxilla is measured and compared for 3 frames. Two frames are used for comparison of resistance to occlusal forces.

Adsorption of plasma proteins on hydrophobic surfaces. IV. Contact angle studies on implanted polymers.

Contact angle studies have been carried out on plasma protein layers adsorbed on selected polymer surfaces under buffered saline at 37 degrees, in an attempt to demonstrate directly a recent suggestion that the interfacial free energy between such protein layers and surrounding liquid phase should be zero at equilibrium. Although an initial contact angle of 180 degrees was always obtained, the angle decayed slowly to a stationary value which varied for any one drop on each polymer surface. The stationary values could be reasonably correlated with the reversible work of adhesion predicted for each polymer/protein combination, suggesting that protein desorption from the solid surface is a dominant event in the contact angle decay process. It is concluded that the data bear more relevance to the protein layer/polymer interface than to the protein layer/solution interface, and that the contact angle technique is not a suitable technique for studying the latter on biomaterials.

A novel technique for studying the adsorption of plasma proteins on hydrophobic surfaces.

A method is shown for deducing the surface area of a pendant drop from the same profile photograph as is normally used to determine interfacial tension. Manipulation of such drops by a micrometer syringe then enables the pendant drop to be used as a surface balance for studying adsorption from bulk solutions. Results are given for the compression of films from solutions of albumin, gamma-globulin, fibrinogen, albumin-gamma-globulin mixture and albumin-gamma-globulin-fibrinogen mixture, and from serum, plasma, and blood at the isooctane-buffer interface. It is suggested that gamma-globulin and fibrinogen films are unfolded at the interface but that albumin films are different in that they consist of an inner, unfolded layer and an outer, globular layer. A film from serum resembles that from gamma-globulin alone, and not that from albumin-gamma-globulin mixture, whereas a film from plasma resembles that of fibrinogen at low compression and that of albumin-gamma-globulin-fibrinogen mixture at high compression. A film from blood is shown to resemble that from plasma.

Adsorption of plasma proteins on hydrophobic surfaces. III. Serum, plasma, and blood.

Liquid-air and liquid-liquid interfaces were used as models for the liquid-solid system of plasma proteins and hydrophobic surfaces in the study of adsorption of serum, plasma, and blood onto these surfaces. The interfacial tension is determined for three phases: air, methylene iodide, and isooctane. Curves of interfacial tension versus time for the various systems are given from which a triple-intersection point, where the protein solution is in equilibrium with each surface, is found. It is shown that albumin, gamma-globulin, and a mixed solution of these at in vivo concentrations behave in characteristic and constant manners at the three interfaces of air, methylene iodide, and isooctane. A range of synthetic surfaces which have constant behavior at equilibrium is deduced and it is concluded that any soft tissue response differences between such surfaces could not be the result of albumin or gamma-globulin.

Adsorption of collagen on model hydrophobic surfaces.

The change in interfacial tension with time for rat-tail tendon collagen adsorbing at the air, methylene iodide and isooctane interfaces has been followed by the pendant drop technique as a model for hydrophobic biomaterials. It is shown that the surface activity of tropocollagen at these three interfaces is less than that of albumin, gamma-globulin, serum of plasma, so that displacement of a preadsorbed plasma protein layer by tropocollagen molecules is energetically unfavourable and unlikely to occur. It is proposed that this may be one reason for the lack of interaction between hydrophobic soft tissue implants and their tissue capsules. It is then speculated that such interaction may require the development of biomaterials with a specific affinity for connective tissue components.

Adsorption of plasma proteins on hydrophobic surfaces. II. Fibrinogen and fibrinogen-containing protein mixtures.

The adsorption of fibrinogen from single solution and from mixed solution with albumin and gamma-globulin has been followed at the air-buffer, isooctane-buffer, and methylene iodide-buffer interfaces by the pendant drop technique. Fibrinogen is shown to form substantial coherent films on isooctane and methylene iodide, suggesting considerable unfolding and lateral association. From this, a novel hypothesis has been proposed to account for the dominance of fibrinogen adsorption from mixed solutions on certain hydrophobic surfaces, in which the spreading pressure of rapidly unfolding fibrinogen molecules is sufficient to desorb other plasma proteins.