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Analysis of mRNA in two haemophilic monozygotic twins offers novel information on the organisation of expressed sequences distal to the coagulation factor VIII gene. These patients show an inversion that, in contrast to the common inversions responsible for 1/5 of all haemophilia A, affects the first rather than intron 22 of the gene. This displaces the most telomeric of the factor VIII exons (exon 1) by approximately 100 kb towards the telomere, and close to the region of the C6.1A gene. This novel inversion creates two hybrid transcription units: one formed by the promoter and first exon of the factor VIII gene followed by a widely expressed sequence; the other by the promoter and coding region of the C6.1A gene plus most of the factor VIII gene (part of intron 1 and exons 2-26). Investigation of this transcription unit reveals that the C6.1A gene has an unsuspected intron in the region coding for the previously described 3'-untranslated tail of the message. Furthermore, exons located beyond the known C6.1A sequence and present in normal transcripts precede exons 2-26 of the factor VIII gene in the hybrid mRNA of the haemophilic twins. The factor VIII sequences in this hybrid mRNA are not expected to be expressed because they lack the first exon, encoding the prepeptide, and follow a translation stop in the C6.1A gene. Leukaemia-related translocations in the C6.1A region suggest that this region may be somewhat unstable.

Almost half of all cases of severe hemophilia A are caused by recurrent DNA inversions, which disrupt the factor VIII (FVIII) gene. These inversions generally occur between a region of intron 22 (int22h) and one of two homologous copies of this region, located 300 to 400 kb telomeric to the FVIII gene. They are routinely detected by a Bcl I Southern blot assay in which the sizes of two of the three normal hybridization bands are characteristically altered. However, atypical hybridization patterns have been observed, and this report describes the first detailed analysis of a hemophilia A patient with such a pattern. The abnormal result was found to be caused by a novel FVIII inversion involving an extra copy of int22h from a site only 70 to 200 kb telomeric of the FVIII gene. Polymerase chain reaction (PCR) allowed one of the inversion junctions to be analyzed, showing that the int22h sequence at this inversion junction was truncated. This patient and his novel inversion provide further evidence that int22h is associated with instability in Xq28.

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.

Two neural networks which are trained on examples of whole words to perform the task of keyword spotting are described. One network is a temporally constrained self-organizing feature map. The other network is a time-delay network which has been modified by the addition of recurrent connections. These networks were tested as secondary processors to a conventional (non-neural network) wordspotter. In this scenario, the conventional system screened incoming speech for potential keywords which are passed to the networks for the final accept/reject determination. The database used for testing contains 20 key words. The test results are summarized in receiver operator characteristic (ROC) curves. These initial results indicate that wordspotting performance is helped by the application of neural network word discriminators as secondary processors. The percentage of keywords recognized was improved at all false alarm rates. >

The application of dynamic neural networks for improving keyword spotter performance is discussed. A conventional wordspotter, which is based on template matching, provides an initial screening of incoming speech for possible keyword occurrences. The role of the neural network is to provide a second layer of discriminant analysis in which the final accept/reject decision is made. In experiments conducted on standard corpora for wordspotting, secondary scoring with temporally constrained Kohonen feature maps resulted in reduced false alarm rates. Preliminary results using a recurrent neural network are also presented. >

The enhancement of coded speech which has been degraded by a tandem connection between wideband and narrowband data links, specifically a delta-modulation (CVSD 16000 b/s) to linear predictive coding (LPC) (2400 b/s) link, is discussed. Speech enhancement is achieved by developing mappings from vector quantization (VQ) codebooks trained on distorted data to VQ codebooks trained on undistorted data. Two different algorithms are adapted to the problem of generating these codebooks. These algorithms are the self-organizing feature map algorithm and learning vector quantization. >

A region of intron 22 of the factor VIII gene, which contains factor VIII-associated gene A (F8A), is repeated twice more nearer the Xq telomere. It has been proposed that intrachromosomal homologous recombination occurs between the intron 22 repeat and either of the two extragenic copies, resulting in the recurrent inversions that cause almost half of all cases of severe haemophilia A. We have precisely defined the repeated region as 9.5 kb of DNA which we have termed int22h (intron 22 homologous region). The junctions of the inversions examined were shown to represent precise exchanges between the int22h repeats, thus providing conclusive evidence for homologous recombination. The three copies of int22h were compared along 8 kb of their length, using chemical mismatch analysis, and found to be 99.9% similar. The presence of such long, almost identical inverted repeats near the Xq telomere could account for the high frequency at which the inversions occur.

Publisher Summary An accurate account of hemophilia and the X-linked nature of the disease that affects males and is transmitted by females were published by Otto in 1803. This description, of course, precedes Mendel's discovery of the laws of inheritance by half a century and that of X-linkage by more than a century. In 1952, the discovery of complementation of the coagulation defect in mixtures of plasma from unrelated patients clearly indicated the existence of two different hemophilias. In the United Kingdom, the two hemophilias were named “classical hemophilia” and “Christmas disease,” while in the United States the terms “hemophilia A” and “hemophilia B” were used. The coagulation factors deficient in hemophilia A and B are now called factors VIII and IX, respectively. The latter is a serine protease, while the former is a cofactor of factor IX; both circulate in the blood in an inactive form. When activated, factors IX and VIII cooperate to cleave and activate factor X, thus together contributing to the proteolytic cascade that controls the conversion of fibrinogen to fibrin. Because hemophilia A and B affect the same blood coagulation step, patients with either type of hemophilia have essentially the same features. These include spontaneous bleeding, especially into joints and muscles, prolonged bleeding from minor cuts, and severe bleeding from lacerations. The expression of cloned genes has led to the introduction of recombinant factor VIII in the treatment of hemophilia A, and the genes for both factors VIII and IX are being used to develop successful approach toward gene therapy.

Surprisingly half of all severe haemophilia A patients have no mutation in the promoter, coding sequences and normal RNA processing signals of the factor VIII gene. Instead they manifest a unique mRNA defect that prevents the amplification of the message across the boundary between exon 22 and 23. This locates the defect to internal regions of intron 22. Novel sequences 3' to exon 22 were isolated from the 9 available patients with the above abnormality by combining RACE and vectorette amplifications on trace amounts of mRNA. This showed that exons 1-22 of the factor VIII mRNA had become part of a hybrid message containing new multi exonic sequences expressed in normal cells. The novel sequences were not located in a YAC covering the whole factor VIII gene. Southern blots from patients probed by novel sequences and clones covering intron 22 showed no obvious abnormalities. This suggested inversions involving intron 22 repeated sequences. Screening of 3 YAC libraries with the novel sequences located them at least 200 kb telomeric (5') to factor VIII and pulsed field gel analysis detected abnormal bands in patients. This demonstrates that the mutations in the patients are inversions of long DNA regions possibly involving the repeated sequences and occurring at the surprising rate of approximately 4 x 10(-6) per gene per gamete per generation.

Haemophilia A is a mutationally heterogeneous disease caused by defects in the large and complex factor VIII gene. Recent studies examining the putative promoter, all exons and most intron/exon boundaries have failed to detect mutations in half the patients with severe disease leading to hypotheses such as mutations in remote controlling regions or even in genes other than factor VIII. We have amplified the factor VIII gene (putative promotor, coding region and polyadenylation/cleavage signal region) in 8 fragments from reverse transcribed mRNA and genomic DNA. Any mutation is then located by chemical mismatch detection and characterised by direct sequencing. This rapid and efficient method has been fully successful and has revealed an unusual cluster of mutations causing severe disease. Of the 28 patients we have reported, 5 had mild or moderate disease and all had a missense mutation. Twenty-three patients were severely affected and 13 of these had different detrimental mutations that were fully characterised at the genomic DNA level. The remaining 10 patients all had mRNA with exon 22 not contiguous to exon 23. Since all exons were normal and so were the splice sites of intron 22, the mutation in these patients should be in the regions of intron 22 that were not screened. These results prove that all haemophilia A cases are due to mutations of the factor VIII gene where, unexpectedly, intron 22 seems to be the target of approximately 40% of the mutations causing severe haemophilia A.

The authors present a neural network which is trained on word examples to perform the wordspotting task. This network has multiple recurrent connections with time delay to account for temporal dynamics. A single network may be trained to recognize one word or many words. A hybrid wordspotter is evaluated in which a conventional wordspotter (based on dynamic time warping word matching) is used to screen incoming speech for potential keywords which are then passed to the network for the final accept/reject decision. Initial tests on a standard wordspotting test corpora resulted in improved keyword recognition at false alarm rates above zero. >

Using an mRNA-based method to examine haemophilia A mutations we provide an explanation for the puzzling report that half of the mutations causing severe disease are not detected by analysis of the putative promoter, exons, and most exon/intron boundaries of the factor VIII gene. An unusual cluster of mutations involving regions of intron 22 not examined earlier leads to defective joining of exons 22 and 23 in the mRNA and caused haemophilia A in 10/24 severely affected UK patients.

Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).