Allelic frequencies and patterns of single-nucleotide polymorphisms in candidate genes for asthma and atopy in Iceland.

Numerous asthma and atopy loci have been reported in studies demonstrating associations of the asthma-related phenotypes atopy, elevated IgE levels, and bronchial hyperresponsiveness with alleles of microsatellite markers and single-nucleotide polymorphisms (SNPs) within specific cytokine/chemokine and IgE-regulating genes. Although the studies reporting these observations are compelling, most of them lack statistical power. We assessed the nature, pattern, and frequency of SNPs in 24 candidate genes in Iceland and looked for associations with asthma and atopy. We identified 42 SNPs with an average minor allele frequency of 20.3% (asthma) and 20.7% (control). Twenty SNPs (48%) were within coding sequences and 90% of those led to a predicted change in protein sequence. No differences were detected in the allelic frequencies of SNPs in any of these candidate genes between control subjects and the patients with atopic asthma. Moreover, linkage analysis that included 269 patients with atopic asthma uncovered no evidence of linkage to markers associated with these genes. We conclude that this study has failed to produce evidence in support of the notion that variations within these 24 candidate atopy and asthma genes significantly influence the expression of the atopic asthmatic phenotype or contribute to the susceptibility of atopic asthma.

Mapping and complex expression pattern of the human NPAP60L nucleoporin gene.

From a clone mapping to human chromosome 22q13.3, we have identified NPAP60L, the human homolog of the rat nuclear pore-associated protein gene, Npap60. The expression pattern of the human copy is much more complex that that of the rat, although conservation of the potential specific function of NPAP60L in male germ cells can be seen for one of the five transcripts. The exon-intron organization of the NPAP60L gene shows the presence of at least three alternate 3' ends, and Northern analysis indicates the possible presence of alternate 5' ends. Somatic cell hybrid mapping revealed additional related copies of NPAP60L on human chromosomes 5, 6, and 14, although it is not known if these are functional genes.

Two novel human RAB genes with near identical sequence each map to a telomere-associated region: the subtelomeric region of 22q13.3 and the ancestral telomere band 2q13.

Two closely related genes have been identified at 2q13 and 22q13.3. These genes show similarity to members of the RAB family of small GTPases. RABL2A and RABL2B differ by three conservative amino acid changes over a total of 228 residues. Both are expressed in all tissues tested. Northern analysis showed that a 2.5-kb transcript is expressed in all tissues tested while a 1.4-kb transcript is specifically expressed only in muscle. The size difference between these two transcripts is the result of differential splicing of an intron within the 3' UTR. RABL2B is located within the subtelomeric region of 22q13.3. RABL2A maps to 2q13, the site of an ancestral telomere fusion event, suggesting that it also may be a subtelomeric gene.

Ring 22 duplication/deletion mosaicism: clinical, cytogenetic, and molecular characterisation.

A patient with several features consistent with duplication of 22q11.2 (cat eye syndrome or CES) was found to be mosaic for a dicentric double ring chromosome 22 on postnatal karyotyping of peripheral blood. The initial karyotype was 46,XX,r(22)(p12q13) [46]/46,XX,dic r(22)(p12q13; p12q13)[4]. The amount of material duplicated in the dic r(22) was determined to include and extend beyond the CES critical region into 22q13.3. However, karyotyping of lymphocytes and fibroblasts, at 27 and 13 months of age respectively, showed no dic r(22) present in any of the cells examined. We suggest that the CES features in this patient, and potentially in other ring cases with CES phenotypic features, might result from a high level of mosaicism for a dic r(22) during early fetal development. Usually this unstable dic r(22) is subsequently lost from most cells.

Discordant measures of androgen-binding kinetics in two mutant androgen receptors causing mild or partial androgen insensitivity, respectively.

We have characterized two different mutations of the human androgen receptor (hAR) found in two unrelated subjects with androgen insensitivity syndrome (AIS): in one, the external genitalia were ambiguous (partial, PAIS); in the other, they were male, but small (mild, MAIS). Single base substitutions have been found in both individuals: E772A in the PAIS subject, and R871G in the MAIS patient. In COS-1 cells transfected with the E772A and R871G hARs, the apparent equilibrium dissociation constants (Kd) for mibolerone (MB) and methyltrienolone are normal. Nonetheless, the mutant hAR from the PAIS subject (E772A) has elevated nonequilibrium dissociation rate constants (k(diss)) for both androgens. In contrast, the MAIS subject's hAR (R871G) has k(diss) values that are apparently normal for MB and methyltrienolone; in addition, the R871G hAR's ability to bind MB resists thermal stress better than the hAR from the PAIS subject. The E772A and R871G hARs, therefore, confer the same pattern of discordant androgen-binding parameters in transfected COS-1 cells as observed previously in the subjects' genital skin fibroblasts. This proves their pathogenicity and correlates with the relative severity of the clinical phenotype. In COS-1 cells transfected with an androgen-responsive reporter gene, trans-activation was 50% of normal in cells containing either mutant hAR. However, mutant hAR-MB binding is unstable during prolonged incubation with MB, whereas normal hAR-MB binding increases. Thus, normal equilibrium dissociation constants alone, as determined by Scatchard analysis, may not be indicative of normal hAR function. An increased k(diss) despite a normal Kd for a given androgen suggests that it not only has increased egress from a mutant ligand-binding pocket, but also increased access to it. This hypothesis has certain implications in terms of the three-dimensional model of the ligand-binding domain of the nuclear receptor superfamily.

Correlation of B-FABP and GFAP expression in malignant glioma.

The murine brain fatty acid binding protein (B-FABP) is encoded by a developmentally regulated gene that is expressed in radial glial cells and immature astrocytes. We have cloned the human B-FABP gene and have mapped it to chromosome 6q22-23. We show that B-FABP mRNA is expressed in human malignant glioma tumor biopsies and in a subset of malignant glioma cell lines, as well as in human fetal retina and brain. Malignant glioma tumors are characterized by cytoplasmic bundles of glial fibrillary acidic protein (GFAP), a protein normally expressed in mature astrocytes. Establishment of malignant glioma cell lines often results in loss of GFAP. The subset of malignant glioma cell lines that express GFAP mRNA also express B-FABP mRNA. Co-localization experiments in cell lines indicate that the same cells produce both GFAP and B-FABP. We suggest that some malignant gliomas may be derived from astrocytic precursor cells which can express proteins that are normally produced at different developmental stages in the astrocytic differentiation pathway.

Characterization of alternative amino acid substitutions at arginine 830 of the androgen receptor that cause complete androgen insensitivity in three families.

We have studied two different missense mutations at arginine-830 in exon 7 of the human androgen receptor (hAR) gene that cause complete androgen insensitivity (CAIS) in three families. These substitutions result from point mutations at nucleotide 2489: a G-->T transversion causes Arg830Leu and a G-->A transition causes Arg830Gln. Genital skin fibroblasts of the patients have negligible androgen-binding capacity. The mutations were recreated in an hAR cDNA expression vector that was transiently transfected into COS-1 cells. Both mutant androgen receptors have increased dissociation rate constants and apparent equilibrium rate constants when measured with 5 alpha-dihydrotestosterone or the synthetic, nonmetabolizable androgens, mibolerone or methyltrienolone. The mutant androgen-binding activities share a distinctive thermal misbehavior. At 37 degrees C R830Q and R830L are about 40% and 10% of normal, respectively. At 22 degrees C both mutants gain androgen binding while the normal decreases by 20%; for R830Q the augmented value approaches 60% of the normal. During prolonged 18 h incubation at 37 degrees C, androgen binding of the normal AR is stable while that of both mutants decreases by at least 85%. Both mutants have a very reduced ability to transactivate a cotransfected androgen-responsive reporter gene, but R830Q is better than R830L. We conclude that arginine-830 is important for A-R complex stability, and that its replacement by glutamine or leucine yields distinctive functional aberrations.(ABSTRACT TRUNCATED AT 250 WORDS)