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.

DNA variation in a 13-Mb region including the F9 gene: inferring the genealogical history and causal role of a hemophilia B mutation (IVS 5+13 A-->G).

About 5.5% of all UK hemophilia B patients have the base substitution IVS 5+13 A-->G as the only change in their factor (F)IX gene (F9). This generates a novel donor splice site which fits the consensus better than the normal intron 5 donor splice. Use of the novel splice site should result in a missense mutation followed by the abnormal addition of four amino acids to the patients' FIX. In order to explain the prevalence of this mutation, its genealogical history is examined. Analysis of restriction fragment length polymorphism in the 21 reference UK individuals (from different families) with the above mutation showed identical haplotypes in 19 while two differed from the rest and from each other. In order to investigate the history of the mutation and to verify that it had occurred independently more than once, the sequence variation in 1.5-kb segments scattered over a 13-Mb region including F9 was examined in 18 patients and 15 controls. This variation was then analyzed with a recently developed Bayesian approach that reconstructs the genealogy of the gene investigated while providing evidence of independent mutations that contribute disconnected branches to the genealogical tree. The method also provides minimum estimates of the age of the mutation inherited by the members of coherent trees. This revealed that 17 or 18 mutant genes descend from a founder who probably lived 450 years ago, while one patient carries an independent mutation. The independent recurrence of the IVS5+13 A-->G mutation strongly supports the conclusion that it is the cause of these patients' mild hemophilia.

Structural elements of the osteopontin SVVYGLR motif important for the interaction with alpha(4) integrins.

The osteopontin SVVYGLR motif binds the integrins alpha(4)beta(1) and alpha(9)beta(1). We show that alpha(4)beta(7) also interacts with this motif and that an SVVYGLR-OH peptide antagonises the alpha(4)beta(7) MAdCAM interaction. The important elements of this motif required to bind alpha(4)beta(1) and alpha(4)beta(7) were probed using a series of mutated peptides based around SVVYGLR. Leu167 is important for the interaction with alpha(4) integrins, as is the C-terminal carboxylic acid of Arg168 exposed by thrombin cleavage. The importance of the acidic group means that SVVYGLR has structural elements in common with other alpha(4) integrin-binding motifs and suggests why thrombin cleavage activates this motif.

The X chromosome and the rate of deleterious mutations in humans.

Monosomy for the X chromosome in humans creates a genetic Achilles' heel for nature to deal with. We report that the human X chromosome appears to have one-third the density of the coding sequence of the autosomes and, because of partial shielding from the high mutation rate of the male sex, that it should also have a lower mutation rate than the autosomes (i.e.,.73). Hence, the X chromosome should contribute one quarter (.33x.73=.24) of the deleterious mutations expected from its DNA content. In this way, selection has possibly moderated risks from mutation in X-linked genes that are thought to have been fixed in their syntenic state since the onset of the mammalian lineage. The unexpected difference in the density of coding sequences indicates that our recent, hemophilia B-based estimate of the rate of deleterious mutations per zygote should be increased from 1.3 to 4 (1.3x3).

Health behaviors of undergraduate African American nursing students.

Health experts describe lifestyle as one of the most important factors influencing health. Adolescents and young adults have been identified as a population that engages in high-risk behaviors. The purposes of this study were to determine the health behaviors of undergraduate African American nursing students and compare the results to findings from studies of other college students. A convenience sample of 214 undergraduate African American nursing students participated in the study. The Health Style: A Self-Test, a Likert-type scale consisting of six behaviors, was used for data collection. Descriptive statistics and analysis of variance were used to analyze the data. Over 80% of the sample had excellent scores for cigarette smoking, alcohol and drug use, and safety behaviors. Over 60% had good scores for nutrition and stress control behaviors. Fifty-one percent of the sample had low scores for exercise and fitness behaviors indicating they are taking unnecessary risk with their health. Compared to other findings, these findings were consistent in all areas except alcohol and drug use. Early identification of at-risk behaviors among nursing students can contribute to the development and implementation of programs by faculty that foster healthy lifestyle behaviors throughout the life span.

Health promotion for urban middle school students: a survey of learning needs.

Community based education programs and community partnerships are crucial for attaining the objectives of Healthy People 2010. The purpose of this descriptive study was to determine the health promotion needs of urban middle school students from the perspective of the participants in a health promotion partnership project. A convenience sample of 161 urban middle school students participated in the study. A Likert scale of health promotion topics was used for data collection. Findings from the study indicated that urban middle school students have a major interest in finding out more about hair care, safety issues, and prevention of infection. Students expressed a need for more information about issues related to why people use drugs, feelings about self, feelings of sadness and worry, and feelings about death. Important variations were found in the priority of interest in topics among the three grades. The researchers concluded that the psychosocial health promotion needs of urban middle school students should be explored further.

Biallelic discrimination assays for the three common Ashkenazi Jewish mutations and a common non-Jewish mutation, in Tay-Sachs disease, using fluorogenic TaqMan probes.

We have developed rapid semiautomated fluorogenic TaqMan assays for the three common Jewish mutations that occur in Tay-Sachs disease, the TATC 4-bp insertion in exon 11 (1,278insTATC), the IVS 12 + 1G --> C, splice site mutation in intron 12 (1421 + 1 G --> C), and the G --> A change at the 3' end of exon 7 (G269S), as well as for a non-Jewish mutation, IVS9 + I G --> A, believed to be prevalent in patients of Celtic descent. The TaqMan assays are designed to run on the ABI SDS 7700 sequence detection system, using allele-specific probes that carry a reporter dye at the 5' end and a quencher dye at the 3' end. Using a 96-well format, all four assays can be performed simultaneously on the same plate, with real-time fluorescence detection or just an end-point plate read. DNA samples from 78 patients identified as carriers by biochemical screening and genotyped by conventional techniques were used to assess the accuracy and efficiency of the probes in allelic discrimination assays. There were no discrepancies noted between previously assigned genotypes and the results obtained by application of this methodology.

Mutation rates in humans. I. Overall and sex-specific rates obtained from a population study of hemophilia B.

A population-based study of hemophilia B mutations was conducted in the United Kingdom in order to construct a national confidential database of mutations and pedigrees to be used for the provision of carrier and prenatal diagnoses based on mutation detection. This allowed the direct estimate of overall (micro), male (v), and female (u) mutation rates for hemophilia B. The values obtained per gamete per generation and the 95% confidence intervals are micro;=7.73 (6. 29-9.12&parr0;x10-6; v=18.8 (14.5-22.9&parr0;x10-6; and u=2.18 (1. 44-3.16&parr0;x10-6. The ratio of male-to-female mutation rates is 8. 64, with a 95% confidence interval of 5.46-14.5. The higher male rate was not caused by a much higher rate of transition at CpG sites in the male. Attempts to detect evidence of gonadal mosaicism for hemophilia B mutation in suitable families did not detect any instances of ovarian mosaicism in any of 47 available opportunities. This suggests that the risk of a noncarrier mother manifesting as a gonadal mosaic by transmitting the mutation to a second child should be <0.062.

Mutation rates in humans. II. Sporadic mutation-specific rates and rate of detrimental human mutations inferred from hemophilia B.

We estimated the rates per base per generation of specific types of mutations, using our direct estimate of the overall mutation rate for hemophilia B and information on the mutations present in the United Kingdom's population as well as those reported year by year in the hemophilia B world database. These rates are as follows: transitions at CpG sites 9.7x10-8, other transitions 7.3x10-9, transversions at CpG sites 5.4x10-9, other transversions 6.9x10-9, and small deletions/insertions causing frameshifts 3.2x10-10. By taking into account the ratio of male to female mutation rates, the above figures were converted into rates appropriate for autosomal DNA-namely, 1.3x10-7, 9.9x10-9, 7.3x10-9, 9.4x10-9, 6.5x10-10, where the latter is the rate for all small deletion/insertion events. Mutation rates were also independently estimated from the sequence divergence observed in randomly chosen sequences from the human and chimpanzee X and Y chromosomes. These estimates were highly compatible with those obtained from hemophilia B and showed higher mutation rates in the male, but they showed no evidence for a significant excess of transitions at CpG sites in the spectrum of Y-sequence divergence relative to that of X-chromosome divergence. Our data suggest an overall mutation rate of 2.14x10-8 per base per generation, or 128 mutations per human zygote. Since the effective target for hemophilia B mutations is only 1.05% of the factor IX gene, the rate of detrimental mutations, per human zygote, suggested by the hemophilia data is approximately 1.3.

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.

Idiopathic intracranial haemorrhage in the fetus.

Intracranial haemorrhage in the fetus has been reported with associated mortality and morbidity. This case report describes idiopathic subdural haematomas diagnosed at 32 weeks of gestation, with delivery by caesarean section of a live male infant in good condition at 34 weeks.

A study of anal intraepithelial neoplasia in HIV positive homosexual men.

OBJECTIVES: To determine the prevalence of high grade anal intraepithelial neoplasia (HGAIN), the value of anal cytology in screening for HGAIN, and the characterisation of epidemiological factors and human papillomavirus (HPV) types. METHODS: Prospective cohort study of HIV positive homosexual men. Subjects were interviewed, underwent STD, anal cytological, and HPV screening at enrolment and at subsequent follow up visits with anoscopy and biopsy at the final visit. 57 enrolled, average CD4 count 273 x 10(6)/l (10-588); 41 completed the cytological surveillance over the follow up period (181 visits, average follow up 17 months), 38 of these had anoscopy and anal biopsy. RESULTS: Oncogenic HPV types were detected in 84% and high grade dyskaryosis in 10.5% (6/57) at enrollment. There was a 70% incidence of high grade dyskaryosis during follow up in patients with negative/warty or low grade dyskaryosis at enrollment. Anoscopy correlated with histology in high grade AIN lesions (sensitivity 91%, specificity 54%) and cytology was 78% sensitive (18/23) for HGAIN on biopsy. CONCLUSIONS: AIN and infection with multiple oncogenic HPV types are very common among immunosuppressed HIV positive homosexual men. Apparent progression from low to high grade cytological changes occurred over a short follow up period, with no cases of carcinoma. All 23 cases of HGAIN were predicted by cytology and/or anoscopy. Future studies focusing on the risk of progression to carcinoma are needed before applying anal cytology as a screening tool for AIN in this population.

Genomic organization and chromosomal localization of the human CUL2 gene and the role of von Hippel-Lindau tumor suppressor-binding protein (CUL2 and VBP1) mutation and loss in renal-cell carcinoma development.

Germline mutations in the von Hippel-Lindau (VHL) disease tumor suppressor gene (TSG) convey a high risk of clear-cell renal-cell carcinoma (CC-RCC) and most sporadic CC-RCCs demonstrate somatic inactivation of the VHL TSG. However, the existence of further CC-RCC gatekeeper genes is implied by CC-RCC kindreds not linked to the VHL gene and the absence of somatic VHL inactivation in approximately 30% of sporadic CC-RCC. Genes that encode proteins which interact with the VHL gene product (VHL) provide candidate gatekeeper RCC genes. VHL forms a multimeric complex with two subunits (B and C) of the SIII (elongin) transcriptional elongation complex and CUL2, a member of the cullin family. Most pathogenic VHL mutations inhibit formation of the VHL/elonginB+C/CUL2 complex. A further VHL-binding protein of unknown function, VBP1, fails to bind to truncated forms of VHL. We have investigated the possible roles of CUL2 and VBP1 in renal tumorigenesis by analyzing sporadic RCC of known VHL mutation or hypermethylation status, including CC-RCC without VHL inactivation (n = 40); CC-RCC with VHL inactivation (n = 35); and non-CC-RCC (n = 14). No VBP1 mutations were identified in 89 sporadic RCCs, suggesting that VBP1 is not an RCC gatekeeper gene. To investigate CUL2, we mapped the CUL2 gene to chromosome band 10p11.1-p11.2, a region reported to show loss of heterozygosity (LOH) in several human cancers (including non-CC-RCC); determined the genomic organization; and performed mutation analysis of the 21 exons identified. Using novel intragenic polymorphisms, we detected LOH in 6/25 informative RCCs; however, no pathogenic CUL2 mutations were identified in the 89 RCCs analyzed. These findings suggest that unless CUL2 is inactivated by epigenetic events, it is not a major RCC TSG. However, CUL2 remains a candidate TSG for other tumor types demonstrating 10p LOH. Genes Chromosomes Cancer 26:20-28, 1999.

Start of UK confidential haemophilia A database: analysis of 142 patients by solid phase fluorescent chemical cleavage of mismatch. Haemophilia Centres.

A national strategy for optimising genetic services in haemophilia A has been initiated in the UK. Solid phase fluorescent chemical cleavage of mismatch is used to screen the entire coding region of factor VIII in six segments: four amplified from the trace of mRNA in blood lymphocytes and two from genomic DNA for the 3.4 kb exon 14 and flanking intron sequences. These segments are analysed in two threefold multiplexes so that the genes of 18 patients can be screened in a single ABI 377 gel. The promoter and polyadenylation signal region are amplified and sequenced directly. We have analysed 142 unrelated patients and identified 141 factor VIII mutations and one Normandy type von Willebrand homozygote. The former mutations include 89 missense, 10 nonsense, 5 frameshift, one 24 bp deletion and one splice signal defect. These comprise 71 different changes, of which 39 have not been previously observed.