Fine mapping and single nucleotide polymorphism association results of candidate genes for asthma and related phenotypes.

Several genome-wide screens for asthma and related phenotypes have been published to date but data on fine-mapping are scarce. For higher resolution we performed a fine-mapping study with 2 cM average spacing in often discussed asthma candidate regions (2p, 5q, 6p, 7p, 9q, 11p, and 12q) to narrow down the regions of interest. All participants of a Caucasian family study (97 families with at least two affected sib pairs) were genotyped for 49 supplementary polymorphic dinucleotide markers. Our results indicate increased evidence for linkage on chromosome 6p, 9q, and 12q. These candidate regions were further analyzed with SNP polymorphisms in the endothelin 1 (EDN1), lymphotoxin alpha (LTA), and neuronal nitric oxide synthase (NOS1) genes. In addition, IL4 -590C>T and IL10 -592C>A, localized on chromosomes 5q and 1q, respectively, have been analyzed for SNP association. Of the six SNPs tested, four revealed weak association with the examined phenotypes. These are the IL10 -592C>A SNP in the interleukin 10 gene (p=0.036 for eosinophil cell counts), the 4124T>C SNP in EDN1 (p=0.044 for asthma), the 3391C>T SNP in NOS1 with eosinophil cell counts (p=0.0086), and the 5266C>T polymorphism, also in the NOS1 gene, for high IgE levels (p=0.022). In summary, fine mapping data enable us to confine asthma candidate regions, while variants of EDN1 and NOS1, or nearby genes, may play an important role in this context.

Particulate air pollution is associated with an acute phase response in men; results from the MONICA-Augsburg Study.

AIMS: Episodes of increased air pollution are associated with increases in hospital admissions for cardiovascular disease. Even modest acute phase responses are associated with increased risk of coronary heart disease. The study investigates whether induction of an acute phase response by exposure to air pollution may contribute to cardiovascular pathology. METHODS AND RESULTS: A prospective cohort study based on a survey in 1984/85 with a 3-year follow-up was conducted in 631 randomly selected men aged 45 to 64 years free of cardiovascular disease at entry 1984/85. Serum C-reactive protein concentrations were determined by a high sensitivity immunoradiometric assay. C-reactive protein concentration was increased in association with the 1985 air pollution episode. In multivariate analyses, elevated concentrations were independently associated with concentrations of total suspended particles and the sulphur dioxide episode. At ambient concentrations of pollution, as noted during the 1985 air pollution episode, the odds of observing C-reactive protein concentrations above 5.7 mg. l(-1)(>90th percentile) tripled, and increases of 26 microg. m(-3)total suspended particles (mean of 5 days) raised the odds of C-reactive protein levels 50% above the 90th percentile. CONCLUSIONS: Exposure to current levels of particulate matter in the atmosphere elicits an acute phase response in randomly selected healthy middle-aged men, which may contribute to the increased cardiovascular risk caused by air pollution.

A genome-wide search for linkage to asthma. German Asthma Genetics Group.

Asthma is among the most frequent chronic diseases in childhood. Although numerous environmental risk factors have already been identified, the basis for familial occurrence of asthma remains unclear. Previous genome screens for atopy in British/Australian families and for asthma in different American populations showed inconsistent results. We report a sib pair study of a sample of 97 families, including 415 persons and 156 sib pairs. Following an extensive clinical evaluation, all participants were genotyped for 351 polymorphic dinucleotide markers. Linkage analysis for asthma identified four chromosomal regions that could to be linked to asthma: chromosome 2 (at marker D2S2298, P = 0.007), chromosome 6 (around D6S291, lowest P = 0.008), chromosome 9 (proximal to D9S1784, P = 0.007), and chromosome 12 (D12S351, P = 0.010). These linkage regions could be reproduced for all loci by analysis of total or specific immunoglobulin E (minimum P values at these regions were 0. 003, 0.001, 0.010, and 0.015, respectively).

Current status of the Asthma and Allergy Database.

The database provides an online resource for access to data on the genetics of asthma and allergy. This report describes the present status of the site. Currently, a detailed description of 88 linkage studies (7164 linkage positions) and 72 mutation studies are available. The results can be accessed in table form or graphically. The database also contains mouse asthma studies and human homology relationships, gene expression studies and links to relevant patents. Technical details about the server architecture, database installation, database construction, database structure and the user interface are explained elsewhere [Wjst and Immervoll (1998) Bioinformatics, 14, 827-828]. The URL is http://cooke.gsf.de

Polymorphism in the murine Tr2-11 gene encoding an orphan receptor, and its exclusion as a candidate gene for the cataract mutation Cat3.

Since the dominant cataract mutation Cat3 was mapped very closely to the murine nuclear receptor TR2-11 gene locus, the corresponding coding region was amplified by PCR using either genomic DNA or eye-derived cDNA of wild-type (C3Hx102)F1 and of homozygous Cat3 cataract animals. The analysis of the complete coding sequences showed no differences. Additionally, the expression level was very similar. Therefore, Tr2-11 was excluded as a candidate for the Cat3 mutation. Surprisingly, the obtained sequences exhibited significant alterations to the murine Tr2-11 sequence reported previously (Lee et al., Genomics 30, 1995, 46-52). The differences in the DNA sequence predict remarkable secondary and tertiary structure alterations of the corresponding protein. The structure model of the new Tr2-11 protein is very similar to related receptors.

Aphakia (ak), a mouse mutation affecting early eye development: fine mapping, consideration of candidate genes and altered Pax6 and Six3 gene expression pattern.

The homozygous mouse mutant aphakia (ak) has been characterized by bilaterally aphakic eyes without a pupil [Varnum DS, Stevens, LC (1968): J Hered 59:147-150]. The mutation was mapped to chromosome 19 [Varnum DS, Stevens, LC (1975): Mouse News Lett 53:35]. Our linkage studies yielded a precise localization of the ak gene 0.6 +/- 0.3 cM proximal to the microsatellite marker D19Mit10 and 0.7 +/- 0.4 cM distal to D19Mit4 and D19Mit91. No recombination was found with the marker D19Mit9 among 418 backcross offspring tested. The developmental control gene Pax2 mapped 11.0 +/- 3.5 cM proximal to ak and is excluded as a candidate gene. Sequence analysis of Fgf8 and Chuk1, which are localized close to the marker D19Mit10, detected no mutations in the ak/ak mutants. Histological analysis of homozygous mutants suggested the arrest of lens development at the lens stalk stage, a transient morphological structure during the formation of the lens vesicle. In the lens remnants, Pax6 and Six3 are expressed, whereas in the persisting lens stalk only Pax6 was detected. The expression pattern of Pax2 appeared normal; Cryaa expression could not be detected. As a consequence of the arrested lens development, other ocular tissues that require for their development information from the intact lens, such as iris, ciliary muscle, retina, and vitreous body, are absent or formed abnormally.

An Internet linkage and mutation database for the complex phenotype asthma.

SUMMARY: The paper presents details of database construction, website installation and server architecture of the asthma and allergy gene database. AVAILABILITY: Database and server templates are available on request from the first author. SUPPLEMENTARY INFORMATION: The URL of the asthma and allergy gene database is http://cooke.gsf.de

Cat3vl and Cat3vao cataract mutations on mouse chromosome 10: phenotypic characterization, linkage studies and analysis of candidate genes.

Cat3vl and Cat3vao are two allelic, dominant cataract mutations that arose independently in the F1 generation after gamma-irradiation of male mice. The cataracts are already present at birth. Examination of the eyes with a slit lamp revealed completely vacuolated lenses in Cat3vl mutants and anteriorly located opacity in Cat3vao mutants. The appearance of the opacities does not differ between the individuals or between heterozygotes and homozygotes. Penetrance of the mutations is complete. Viability and fertility of the mutants are normal except in the case of the Cat3vl homozygotes. Cat3vao was assigned to the distal part of mouse chromosome 10, 3.2 +/- 0.9 cM away from the visible marker Steel (SlgbH). Using polymorphic markers the following locus order was found: D10Mit230-(0.2 +/- 0.1 cM)-Cat3vao-(2.5 +/- 0.6 cM)-D10Mit70. No recombinants were found between Cat3vao and the markers D10Mit4l and D10Mit95 among 921 offspring. The results exclude allelism of Cat3vao with CatLop or To2, which also map to chromosome 10. Candidate genes were tested by examination of their expression in the eye of newborn mice and by analysis of cDNA sequences. So far, negative results have been obtained for the genes encoding the proteoglycans lumican and decorin, the nuclear orphan receptor Tr2-11 and the transcription factor Elk3. Based on syntenic homology of the Cat3 region to the human chromosome 12q, the Cat3 mutants are discussed as mouse models for cornea plana congenita in man. The recovery of the Cat3 mutations demonstrates the importance of the corresponding locus for proper eye development.

Protein O-glycosylation in Saccharomyces cerevisiae: the protein O-mannosyltransferases Pmt1p and Pmt2p function as heterodimer.

The protein O-mannosyltransferases Pmt1p and Pmt2p are catalyzing the O-glycosylation of serine and threonine residues in the endoplasmic reticulum of yeast. Deletion of each of these proteins by disruption of the corresponding gene leads to a dramatic decrease of mannosyltransferase activity in vitro. With an anti-Pmt1p immunoaffinity column a complex of Pmt1p and a second protein was purified; this protein turned out to be Pmt2p. Overexpression of Pmt1p or Pmt2p, respectively, does not increase mannosyltransferase activity in vitro. Overexpression of both mannosyltransferases together, however, raises in vitro activity threefold. These data indicate that Pmt1p and Pmt2p function as a complex catalyzing protein O-glycosylation in yeast.

PMT3 and PMT4, two new members of the protein-O-mannosyltransferase gene family of Saccharomyces cerevisiae.

Two genes PMT3 and PMT4 were identified by polymerase chain reaction of genomic DNA using primers derived from regions of high homology between the products of three genes PMT1, PMT2 of Saccharomyces cerevisiae and part of a PMT1 related sequence of Kluyveromyces lactis. Pmt1p and Pmt2p are mannosyltransferases involved in the transfer of a mannosyl residue from dolichyl phosphate-D-mannose (Dol-P-Man) to seryl and threonyl residues in proteins. The products encoded by the PMT3 and PMT4 genes have almost identical hydropathy profiles in comparison to PMT1 and PMT2: a hydrophobic N- and C-terminal third each with multiple potential transmembrane helices and a central hydrophilic part. The predicted Pmt3p contains 753 amino acids, four potential N-glycosylation sites and it is significantly homologous to Pmt1p, Pmt2p and Pmt4p. Pmt4p contains 762 amino acids and two potential N-glycosylation sites. Northern blot analysis showed a single mRNA transcript of PMT3 and PMT4 of 2.8 kb. Thus PMT3 and PMT4 are two new members of the PMT gene family. The pmt4 null mutant the pmt3 pmt4 double null mutant, but not pmt3 null mutant, showed a significant shift of chitinase due to under glycosylation of the enzyme. The triple disruption pmt2 pmt3 pmt4 and the quadruple disruption result in a lethal phenotype.

Fungal glycoproteins and their biosynthetic pathway as potential targets for antifungal agents.

The yeast cell wall as a good antifungal target is discussed in general. More specifically the reaction, catalyzed by Dol-P-Man: protein O-D-mannosyltransferase is proposed as a new potential target. Six genes responsible for this endoplasmic reticulum-localized reaction have been cloned and characterized so far. Triple disruptions of these genes are either lethal or the corresponding cells have to be osmotically stabilized to survive. No inhibitors of this reaction are as yet known.

PMT1, the gene for a key enzyme of protein O-glycosylation in Saccharomyces cerevisiae.

The integral endoplasmic reticulum membrane protein catalyzing the initial reaction of protein O-glycosylation in Saccharomyces cerevisiae has been purified to homogeneity. The 92-kDa N-glycosylated protein transfers mannose residues from dolichyl phosphate-D-mannose to specific serine/threonine residues of proteins entering the secretory pathway. This type of mannosyl transfer reaction has so far been observed only in fungal cells. Oligonucleotides derived from peptide sequences of the transferase were used to screen a genomic yeast library. A clone was isolated which contains an open reading frame of 2451 bp corresponding to an mRNA transcript of 3 kb. The predicted protein consists of 817 amino acids including three potential N-glycosylation sites. The hydropathy plot indicates a tripartite structure of the protein: an amino-terminal third and a carboxyl-terminal third, both with multiple potential transmembrane helices, and a central hydrophilic part. Expression of the clone in Escherichia coli resulted in mannosyltransferase activity. Gene disruption led to a complete loss of in vitro mannosyltransferase activity from dolichyl phosphate-D-mannose to a peptide used as acceptor in the enzymatic assay. In vivo it was observed, however, that protein O-mannosylation in the disruptant had decreased only to about 40-50%, indicating the existence of an additional transferase which had not been measured by the in vitro enzyme assay.