The APCI1307K allele and cancer risk in a community-based study of Ashkenazi Jews.

Mutations in APC are classically associated with familial adenomatous polyposis (FAP), a highly penetrant autosomal dominant disorder characterized by multiple intestinal polyps and, without surgical intervention, the development of colorectal cancer (CRC). APC is a tumour-suppressor gene, and somatic loss occurs in tumours. The germline T-to-A transversion responsible for the APC I1307K allele converts the wild-type sequence to a homopolymer tract (A8) that is genetically unstable and prone to somatic mutation. The I1307K allele was found in 6.1% of unselected Ashkenazi Jews and higher proportions of Ashkenazim with family or personal histories of CRC (ref. 2). To evaluate the role of I1307K in cancer, we genotyped 5,081 Ashkenazi volunteers in a community survey. Risk of developing colorectal, breast and other cancers were compared between genotyped I1307K carriers and non-carriers and their first-degree relatives.

A variety of genetic mechanisms are associated with the Prader-Willi syndrome.

An extensive set of chromosome 15 DNA polymorphisms and densitometric analysis with four markers mapping to the Prader-Willi chromosome region (PWCR) of chromosome 15 have been used to characterize a cohort of 30 subjects with classical Prader-Willi syndrome (PWS). Molecular analysis enabled the classification of the PWS subjects into four groups: (A) 18 subjects (60%) had deletions of paternal 15q11-13 involving a common set of DNA markers. Two subjects had differently sized deletions, one larger and one smaller than the other cases. (B) Eight (27%) had maternal uniparental disomy for chromosome 15. (C) One (3%) had a marker chromosome carrying an extra copy of the PWCR. The marker chromosome was demonstrated to be of paternal origin and the two intact chromosomes were maternally derived. This case represents an apparent exception to the generally held view that PWS is associated with an absence of paternally inherited gene(s) located in the PWCR. (D) The remaining three cases (10%) had none of the above abnormalities. This last subgroup of patients has not previously been well characterized but could represent limited deletions not detectable with the markers used or abnormalities in the imprinting process. These cases represent potentially valuable resources to elucidate more precisely the fundamental disorders responsible for PWS.

Stereospecific inversion of (R)-(-)-benoxaprofen in rat and man.

(R)-(-)-benoxaprofen is stereospecifically inverted to the (S)-(+)-enantiomer by rats and humans. The rate of inversion is much faster in rats (t 1/2 ca. 2.5 h) than in humans (t 1/2 108 h). Inversion in rats apparently does not occur in the liver, but can be brought about in vitro by an everted intestinal sac preparation, suggesting that the transformation takes place while passing through the gut wall.

The pharmacokinetics and acceptability of benoxaprofen following rectal administration.

The bioavailability of a single 300 mg dose of benoxaprofen was compared after rectal and oral administration in 5 subjects. The total absorption rectally from a suppository was 83% of that achieved orally from a capsule. Six further subjects took a 300 mg benoxaprofen suppository twice daily for 12 d. Steady state plasma levels (mean level 94 microgram/ml) were reached at about 120 h, while the elimination half-life for benoxaprofen was approximately 38 h in this study. The suppositories were well tolerated. The plasma levels obtained compared very favorably with theoretically expected levels and levels obtained after comparable oral doses.

Determination of isamoxole (N-butyl-N-(4-methyloxazol-2-yl)-2-methylpropanamide, LRCL 3950) in plasma by selected ion monitoring using a stable isotope internal standard.

A selected ion monitoring method for the estimation of an anti-allergy compound isamaxole (N-butyl-N-(4-methyloxazol-2-yl)-2-methylpropanamide, LRCL 3950) in plasma has been developed. The method uses a stable analogue containing nine deuterium atoms as internal standard. The assay is sensitive to 2 ng ml-1 plasma and has been used to examine plasma levels of isamoxole after doses of the compound to animals and man.

Determination of benoxaprofen [2-(4-chlorophenyl)-alpha-methyl-5-benzoxazoleacetic acid, LRCL 3794] in biological fluids.

Benoxaprofen, a novel anti-inflammatory compound, is efficiently (greater than 95%) extracted from plasma and urine in the pH range 1 to 5 into either chloroform or ether. The compound is determined either by UV spectroscopy or by gas-liquid chromatography of the methyl ester (formed by reaction with diazomethane) on a column of 3% of silicone gum E-301 on DCMS-treated Chromosorb W, with detection by flame ionisation (limit 0.3 microgram/ml) or electron capture (limit 0.01 microgram/ml). For rapid routine use, the UV method (limit ca. 5 microgram/ml) gives good agreement with the specific methods.

Novel patterns of inheritance of genetic disease are illustrated by the Angelman syndrome.

OBJECTIVE: To characterise the molecular abnormalities present in a cohort of patients with the Angelman syndrome. METHODS: DNA samples from 10 patients with the Angelman syndrome were investigated with molecular probes. Family studies were performed by means of DNA polymorphism analysis and densitometric estimation of allele copy number to determine the underlying mutation and its parental origin. RESULTS: Nine probands were shown to have molecular (DNA) deletions involving chromosome 15q11-q13. Polymorphism analyses demonstrated that all deletions were maternal in origin. Five of the nine had normal karyotypes, with deletions only detected after DNA study. One patient had inherited both chromosomes 15 from her father. This represented an example of paternal uniparental disomy of chromosome 15. CONCLUSIONS: Development of the Angelman syndrome can result from either deletion of the maternally-derived copy of chromosome 15q11-q13 or the presence of two paternally derived copies of chromosome 15, that is, uniparental disomy. DNA testing allows the identification of deletions that are not seen on cytogenetic analysis and can provide additional information regarding the parental origin of the deletion. Uniparental disomy is most readily established by DNA studies.

Comparison of high resolution cytogenetics, fluorescence in situ hybridisation, and DNA studies to validate the diagnosis of Prader-Willi and Angelman's syndromes.

Eighty seven referrals with Prader-Willi syndrome and 49 with Angelman's syndrome were studied. High resolution cytogenetics was performed on all probands. Molecular studies, performed on the proband and both parents in each case, utilised multiple probes from within and distal to the 15(q11-13) region in order to establish the presence of DNA deletion or uniparental disomy. In addition, FISH, with probes at D15S11 and GABR beta 3 from the Prader-Willi syndrome/Angelman's syndrome region, was performed on a subset of 25 of these patients. In the referral group with Prader-Willi syndrome, 62 patients had a normal karyotype and 25 were deleted on high resolution cytogenetics. Twenty nine were found to be deleted with DNA techniques. In the Angelman's syndrome group, 37 had a normal karyotype and 12 were deleted on high resolution cytogenetics while 26 were deleted on molecular studies. The diagnosis was reassessed in 35 referrals with Prader-Willi syndrome and 11 with Angelman's syndrome following a non-deleted, non-disomic result. Of individuals who were neither deleted nor disomic on DNA studies, a false positive rate of 11.4% (4/35) for Prader-Willi syndrome and 16.7% (2/12) for Angelman's syndrome was found for a cytogenetically detected deletion. The false negative rate for deletion detected on high resolution cytogenetics was 19.5% (12/62) for Prader-Willi syndrome and 35% (13/37) for Angelman's syndrome. Thus high resolution cytogenetics was shown to be unreliable for deletion detection and should not be used alone to diagnose either syndrome. There were no discrepancies with FISH in 25 cases when FISH was compared with the DNA results, indicating that FISH can be used reliably for deletion detection in both syndromes.

Familial unbalanced translocation t(8;15)(p23.3;q11) with uniparental disomy in Angelman syndrome.

A 29-year-old male with Angelman syndrome and an unbalanced reciprocal translocation, 45,XY,-8, -15, +der(8),t(8;15)(p23.3;q11)pat, was evaluated with DNA studies. These showed the underlying mechanism to be paternal uniparental disomy. This is the second case reported of Angelman syndrome that has resulted from a familial unbalanced reciprocal translocation.

Physical mapping studies at D15S10: implications for candidate gene identification in the Angelman syndrome/Prader-Willi syndrome chromosome region of 15q11-q13.

The Angelman syndrome (AS) and Prader-Willi syndrome (PWS) loci have been mapped to chromosome 15q11-q13. Chromosomal deletions of differing parental origin in the two syndromes have been interpreted as being due to genetic imprinting. Molecular analysis of patients with varying deletions has localized the AS locus to the interval between D15S113 and GABRB3 and the PWS locus between D15S13 and D15S113. In the present study, DNA cloning and physical mapping techniques have been used to characterize the AS/PWS chromosome region in the vicinity of D15S10, a locus that is telomeric to D15S113 and centromeric to GABRB3. A CpG island near TD3-21 at D15S10 has been cloned, allowing the identification of a widely expressed 4.5-kb transcript and providing a novel DNA marker, OP3, at this locus. OP3 and TD3-21 have been used to construct a long-range physical map extending over approximately 2800 kb. Clusters of rare-cutting restriction sites on this map locate four other CpG islands. Since these CpG islands lie within the minimum deletion intervals for AS and PWS, they mark the possible locations of candidate genes for the two syndromes.

Characterization of the CHD family of proteins.

The murine gene CHD1 (MmCHD1) was previously isolated in a search for proteins that bound a DNA promoter element. The presence of chromo (chromatin organization modifier) domains and an SNF2-related helicase/ATPase domain led to speculation that this gene regulated chromatin structure or gene transcription. This study describes the cloning and characterization of three novel human genes related to MmCHD1. Examination of sequence databases produced several more related genes, most of which were not known to be similar to MmCHD1, yielding a total of 12 highly conserved CHD genes from organisms as diverse as yeast and mammals. The major region of sequence variation is in the C-terminal part of the protein, a region with DNA-binding activity in MmCHD1. Targeted deletion of ScCHD1, the sole Saccharomyces cerevesiae CHD gene, was performed with deletion strains being less sensitive than wild type to the cytotoxic effect of 6-azauracil. This finding suggested that enhanced transcriptional arrest at RNA polymerase II pause sites due to 6-azauracil-induced nucleotide pool depletion was reduced in the deletion strain and that ScCHD1 inhibited transcription. This observation, along with the known roles of other proteins with chromo or SNF2-related helicase/ATPase domains, suggests that alteration of gene expression by CHD genes might occur by modifications of chromatin structure, with altered access of the transcriptional apparatus to its chromosomal DNA template.

Fluorescence in-situ hybridisation and molecular studies used in the characterisation of a Robertsonian translocation (13q15q) in Prader-Willi syndrome.

A patient with classical Prader-Willi syndrome was found to have a Robertsonian translocation 45,XY,t(13q15q)mat. On CBG banding, the translocation chromosome had a large centromere with one primary constriction. Using fluorescence in situ hybridisation, positive signals were obtained with chromosome 13 and chromosome 15 centromere probes, proving that the translocation was dicentric. NOR banding was negative in this chromosome, suggesting that the breakpoints were at 13p11 and 15p11. DNA studies showed that, while there was no deletion involving 15(q11-13), maternal uniparental disomy for chromosome 15 was present. We compare our findings with the five other cases of familial Robertsonian translocation PWS that have been reported.

The metabolism of isamoxole in the rat and guinea-pig.

1. The absorption, metabolism and excretion of isamoxole, (2-methyl-N-butyl-N-(4-methyloxazol-2-yl)propanamide), a compound with anti-allergy properties, has been studied in the rat and guinea-pig. 2. The compound was well-absorbed by both species after oral doses of 50 to 150 mg/kg. It underwent extensive first-pass metabolism in the liver, and was excreted as a mixture of metabolites, predominantly in the urine, within 48 h. 3. Three major routes of metabolism were involved, namely deacylation, oxidative ring scission and alkyl oxidation. 4. A major plasma and urine metabolite was 1-butyl-3-(1-carboxyethyl)urea, and this was accompanied by low levels of its cyclized product 3-butyl-5-methylhydantoin.

Evolution of the avian sex chromosomes from an ancestral pair of autosomes.

Among the mechanisms whereby sex is determined in animals, chromosomal sex determination is found in a wide variety of distant taxa. The widespread but not ubiquitous occurrence, not even within lineages, of chromosomal sex determination suggests that sex chromosomes have evolved independently several times during animal radiation, but firm evidence for this is lacking. The most favored model for this process is gradual differentiation of ancestral pairs of autosomes. As known for mammals, sex chromosomes may have a very ancient origin, and it has even been speculated that the sex chromosomes of mammals and birds would share a common chromosomal ancestry. In this study we showed that the two genes, ATP5A1 and CHD1, so far assigned to the female-specific W chromosome of birds both exist in a very closely related copy on the Z chromosome but are not pseudoautosomal. This indicates a common ancestry of the two sex chromosomes, consistent with the evolution from a pair of autosomes. Comparative mapping demonstrates, however, that ATP5A1 and CHD1 are not sex-linked among eutherian mammals; this is also not the case for the majority of other genes so far assigned to the avian Z chromosome. Our results suggest that the evolution of sex chromosomes has occurred independently in mammals and birds.

Bloom syndrome and maternal uniparental disomy for chromosome 15.

Bloom syndrome (BS) is an autosomal recessive disorder characterized by increases in the frequency of sister-chromatid exchange and in the incidence of malignancy. Chromosome-transfer studies have shown the BS locus to map to chromosome 15q. This report describes a subject with features of both BS and Prader-Willi syndrome (PWS). Molecular analysis showed maternal uniparental disomy for chromosome 15. Meiotic recombination between the two disomic chromosomes 15 has resulted in heterodisomy for proximal 15q and isodisomy for distal 15q. In this individual BS is probably due to homozygosity for a gene that is telomeric to D15S95 (15q25), rather than to genetic imprinting, the mechanism responsible for the development of PWS. This report represents the first application of disomy analysis to the regional localization of a disease gene. This strategy promises to be useful in the genetic mapping of other uncommon autosomal recessive conditions.