Histamine sustains fMLP-stimulated leukocyte adhesion in rat mesenteric venules in vivo

Hereditary non-syndromic profound deafness affects about 1 in 2000 children prior to language acquisition. In 80% of the cases, the mode of transmission is autosomal recessive. The number of genes involved in these recessive forms of isolated deafness (DFNB genes) has been estimated to between 30 and 100. So far, ten DFNB genes have been mapped to human chromosomes, one of which has been isolated. By linkage analysis of a single family whose members were affected with profound deafness, some of them presenting with vestibular dysfunction, DFNB2 has been mapped to chromosome 11q13 (ref. 3). The gene responsible for a form of Usher syndrome type I, USH1B, has been assigned to the same chromosomal region. Usher syndrome associates profound congenital deafness and vestibular dysfunction with retinitis pigmentosa. In the homologous murine region are located the shaker-1 mutations responsible for deafness and vestibular dysfunction. It has been demonstrated that the murine shaker-1 and human USH1B phenotypes result from mutations in the gene encoding myosin-VIIA. Based on mapping data as well as on the similarities between the phenotypes of DFNB2-affected patients and shaker-1 mouse mutants, we have proposed that a defective myosin-VIIA may also be responsible for DFNB2 (ref. 1). Sequence analysis of each of the coding exons of the myosin-VIIA gene (MYO7A) was thus undertaken in the DFNB2-affected family. In the last nucleotide of exon 15, a G to A transition was detected, a type of mutation that is known to decrease the efficiency of splicing. Accordingly, this result shows that different mutations in MYO7A result in either an isolated or a syndromic form of deafness.     

Localization of the Usher syndrome type ID gene (Ush1D) to chromosome 10

The Usher syndromes (USH) are a group of autosomal recessive diseases characterized by progressive pigmentary retinopathy and sensorineural hearing loss. Five USH genes have been mapped and at least one additional gene is known to exist. By homozygosity mapping in a consanguineous family, a sixth USH gene has been localized. Clinical findings in the four affected children are consistent with established diagnostic criteria for Ush1. Linkage to known USH loci was excluded, and using two genomic DNA pools, one from the affected children and the other from the parents, 161 polymorphic markers evenly spaced across the autosomal human genome were screened. The location of the Ush1D gene was defined by the only region showing homozygosity by descent in the affected siblings, a 15 cM interval on chromosome 10q bounded by D10S529 and D10S573.     

HIV-1 viral load: comparative evaluation of three commercially available assays in Argentina

Autosomal recessive nonsyndromic hearing loss (ARNSHL) is the most common form of hereditary hearing impairment (HHI). To date, 16 different loci have been reported, making ARNSHL an extremely heterogeneous disorder. One of these loci, DFNB4, was mapped to a 5-cM interval of 7q31 in a large Middle-Eastern Druze family. This interval also includes the gene for Pendred syndrome. We report on three new families with HHI from the Madras region of southern India that demonstrate linkage to 7q. Their pedigrees are compatible with autosomal recessive inheritance. Furthermore, the largest family identifies a novel locus (DFNB17) telomeric to the DFNB4 and Pendred intervals. A 3-cM region of homozygosity by descent between markers D7S486 and D7S2529 is present in all affected individuals in this family and generates a multipoint LOD score of 4.24. The two other families map to the previously reported DFNB4 region but have insufficient power to attain significant LOD scores. However, mutations in the Pendred syndrome gene are present in one of these families.     

Connexin26 mutations associated with the most common form of non-syndromic neurosensory autosomal recessive deafness (DFNB1) in Mediterraneans

Non-syndromic neurosensory autosomal recessive deafness (NSRD) is the most common form of genetic hearing loss. Previous studies defined at least 15 human NSRD loci. Recently we demonstrated that DFNB1, located on the long arm of chromosome 13, accounts for approximately 80% of cases in the Mediterranean area. Further analysis with additional markers now identifies several recombinants which narrow the candidate region to approximately 5 cM, encompassed by markers D13S141 and D13S232 and including several ESTs and candidate genes, including the connexin26 (GJB2) gene. Analysis of PCR products from our affected patients' DNA shows two frameshift mutations in the connexin26 gene. Deletion of a G within a stretch of six Gs at position 35 of the GJB2 cDNA (mutation 35delG) leads to premature chain termination and is present in 63% of NSRD chromosomes, demonstrating linkage to chromosome 13. Deletion of a T at position 167 of GJB2 (mutation 167delT), also resulting in premature chain termination, was detected in another patient. Four neutral sequence polymorphisms were also identified. These findings are in agreement with a recent study showing that mutations in the connexin26 gene are associated with genetic forms of deafness in three Pakistani families and that GJB2 is DFNB1. Connexin26 is a member of a large family of proteins involved in formation of gap junctions, which are involved in electrical synapses and the direct transfer of small molecules and ionic currents between neighboring cells. The identification of GJB2 as the DFNB1 gene should provide a better understanding of the biology of normal and abnormal hearing, help form the basis for diagnosis and may facilitate development of strategies for treatment of this common genetic disorder.     

The comparative effects of the NOS inhibitor, Nomega-nitro-L-arginine, and the haemoxygenase inhibitor, zinc protoporphyrin IX, on tumour blood flow

The present study was undertaken to analyse the loss of heterozygosity (LOH) of the three genes, BRCA1, BRCA2 and ATM, and their correlation to clinicopathological parameters in sporadic breast cancer. We studied 59 sets of invasive ductal carcinoma, compared to matched normal control DNA. Microsatellite markers intragenic to BRCA1 (D17S1323, D17S1322, D17S855), BRCA2 (D13S1699, D13S1701, D13S1695) and ATM (D11S2179) were simultaneously used. In addition, one marker telomeric to BRCA2 (D13S1694) and four markers flanking ATM were analysed (D11S1816, D11S1819, D11S1294, D11S1818). Thirty-one per cent of the informative cases showed loss of heterozygosity for the BRCA1 gene, 22.8% for BRCA2 gene and 40% for ATM. LOH of BRCA1 correlated with high grade tumors (p=0.0005) and negative hormone receptors (p=0.01). LOH of ATM correlated with higher grade (p=0.03) and a younger age at diagnosis (p=0.03) in our set of tumors. No correlations were detected between BRCA2 LOH and any of the analysed clinicopathological parameters. However, a correlation was detected between allelic loss of the D13S1694 marker, telomeric to BRCA2, and larger tumor sizes and negative estrogen receptors, favoring the hypothesis of the presence of another putative tumor suppressor gene, telomeric to BRCA2, in the 13q12-q14 region. Only 11 tumors had LOH at more than one of the three genes, most of them (6/11) associated LOH of BRCA1 and ATM. One tumor only combined loss of the three genes BRCA1, BRCA2 and ATM.     

An alpha-tectorin gene defect causes a newly identified autosomal recessive form of sensorineural pre-lingual non-syndromic deafness, DFNB21

In our efforts to identify new loci responsible for non-syndromic autosomal recessive forms of deafness, DFNB loci, we have pursued the analysis of large consanguineous affected families living in geographically isolated areas. Here, we report on the study of a Lebanese family comprising nine members presenting with a pre-lingual severe to profound sensorineural isolated form of deafness. Linkage analysis led to the characterization of a new locus, DFNB21, which was assigned to chromosome 11q23-25. Already mapped to this chromosomal region was TECTA. This gene encodes alpha-tectorin, a 2155 amino acid protein which is a component of the tectorial membrane. This gene recently has been shown to be responsible for a dominant form of deafness, DFNA8/12. Sequence analysis of the TECTA gene in the DFNB21-affected family revealed a G to A transition in the donor splice site (GT) of intron 9, predicted to lead to a truncated protein of 971 amino acids. This establishes that alpha-tectorin mutations can be responsible for both dominant and recessive forms of deafness. Comparison of the phenotype of the DFNB21 heterozygous carriers with that of DFNA8/12-affected individuals supports the hypothesis that the TECTA mutations which cause the dominant form of deafness have a dominant-negative effect. The present results provide genetic evidence for alpha-tectorin forming homo- or heteromeric structures.     

Refined genetic mapping of autosomal dominant retinitis pigmentosa locus RP18 reduces the critical region to 2 cM between D1S442 and D1S2858 on chromosome 1q

Linkage analysis was performed on a large Danish family to refine the position of RP18, the locus for autosomal dominant retinitis pigmentosa, mapped previously between D1S534 and D1S305 in chromosome 1p13-q21. We genotyped the family members for five microsatellite-type DNA polymorphisms and mapped RP18 between D1S422 and D1S2858 to a region of less than 2 cM. No obvious candidate gene has yet been assigned to the chromosomal interval defined here.     

A gene for autosomal dominant hypohidrotic ectodermal dysplasia (EDA3) maps to chromosome 2q11-q13

Autosomal dominant hypohidrotic ectodermal dysplasia (ADHED) is a disorder characterized by fine, slow-growing scalp and body hair, sparse eyebrows and eyelashes, decreased sweating, hypodontia, and nail anomalies. By genetic linkage analysis of a large ADHED kindred, we have mapped a gene for ADHED (EDA3) to the proximal long arm of chromosome 2 (q11-q13). Obligate recombinations localize EDA3 to an approximately 9-cM interval between D2S1321 and D2S308, with no apparent recombinations with markers D2S1343, D2S436, D2S293, D2S1894, D2S1784, D2S1890, D2S274, and CHLC.GAAT11C03.     

Methylphenidate: diurnal effects on locomotor and stereotypic behavior in the rat

The Usher syndromes (US) are a group of inherited disorders that feature autosomal recessive neurosensory hearing loss or deafness with retinitis pigmentosa (RP). Moderate to severe non-progressive high frequency hearing loss with RP and normal vestibular function describes Usher syndrome type IIa, which has been localized to 1q41. Severe retinal degeneration in the inbred mouse strain RBF/DnJ is caused by rd3, a recessive gene located on mouse chromosome 1 distal to akp1 in a region which is orthologous to human 1q32-q42. We evaluated rd3 as a candidate for orthology with USH2A by first reducing and refining the relatively broad region in which rd3 is thought to reside. DNA of offspring from an RBF/DnJ x MOLF/Ei backcross was genotyped with PCR markers closely flanking the predicted location of rd3. Our haplotype analysis re-positioned rd3 to a 3.6 cM region between markers D1Mit273 (cen) and D1Mit209 (tel), consistent with the expected position of an USH2A murine orthologue. Consequently, rd3 is a positional candidate for Usher type IIa. Next we assessed the rd3/rd3 audiological phenotype to see how closely it paralleled that of Usher IIa. Audiological evaluation of mice at various ages revealed evidence of high frequency progressive hearing loss, previously unreported in the RBF/DnJ strain. However, this newly discovered hearing deficit was observed to be inherited independently of rd3, establishing that a completely different gene is responsible.     

Search for a founder mutation in idiopathic focal dystonia from Northern Germany

Both the discovery of the DYT1 gene on chromosome 9q34 in autosomal dominant early-onset torsion dystonia and the detection of linkage for one form of adult-onset focal dystonia to chromosome 18p (DYT7) in a family from northern Germany provide the opportunity to further investigate genetic factors in the focal dystonias. Additionally, reports of linkage disequilibrium between several chromosome 18 markers and focal dystonia, both in sporadic patients from northern Germany and in members of affected families from central Europe suggest the existence of a founder mutation underlying focal dystonia in this population. To evaluate the role of these loci in focal dystonia, we tested 85 patients from northern Germany who had primary focal dystonia, both for the GAG deletion in the DYT1 gene on chromosome 9q34 and for linkage disequilibrium at the chromosome 18p markers D18S1105, D18S1098, D18S481, and D18S54. None of these patients had the GAG deletion in the DYT1 gene. Furthermore, Hardy-Weinberg analysis of markers on 18p in our patient population and in 85 control subjects from the same region did not support linkage disequilibrium. Taken together, these results suggest that most cases of focal dystonia in patients of northern German or central European origin are due neither to the GAG deletion in DYT1 nor to a proposed founder mutation on chromosome 18p but must be caused by other genetic or environmental factors.     

Genetic diversity at six short tandem repeat loci within the state of Victoria, Australia

A new multiplex PCR system, developed by the Forensic Science Service (FSS) in the United Kingdom, permits the coamplification and typing of six short tandem repeat (STR) loci: HUMFGA, D8S1179, HUMTHO1, HUMvWA, D18S51, D21S11 and the sex determining marker Amelogenin. Data are presented on these six STRs for two populations in the state of Victoria, Australia: Caucasian and Asian. Whilst several worldwide databases are already available for the STR loci HUMTHO1 and HUMvWA, only relatively few databases exist for D8S1179, D18S51, D21S11 and HUMFGA. Allele frequencies at each locus displayed some fluctuations between the two populations. This is particularly so for HUMTHO1. Generally, however, the most common allele at each locus was the same in all populations, at all loci. A novel D8S1179 allele was found in Asians, provisionally identified as allele 19. Results for the six loci were compared with similar data from three UK resident populations: Caucasian, Afro-Caribbean and Asian (Indian/Pakistani) populations. These indicated that ethnically similar populations display similar allele frequencies, while the Australian Asian and UK Afro-Caribbean were found to be distinct.     

Mutation of a gene encoding a protein with extracellular matrix motifs in Usher syndrome type IIa

Usher syndrome type IIa (OMIM 276901), an autosomal recessive disorder characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa, maps to the long arm of human chromosome 1q41 between markers AFM268ZD1 and AFM144XF2. Three biologically important mutations in Usher syndrome type IIa patients were identified in a gene (USH2A) isolated from this critical region. The USH2A gene encodes a protein with a predicted size of 171.5 kilodaltons that has laminin epidermal growth factor and fibronectin type III motifs; these motifs are most commonly observed in proteins comprising components of the basal lamina and extracellular matrixes and in cell adhesion molecules.     

Isolation of eukaryotic ribosomal proteins. Purification and characterization of the 40 S ribosomal subunit proteins Sa, Sc, S3a, S3b, S5', S9, S10, S11, S12, S14, S15, S15', S16, S17, S18, S19, S20, S21, S26, S27', and S29

The proteins of the small subunit of rat liver ribosomes were separated into five main groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. Twenty-one proteins (Sa, Sc, S3a, S3b, S5', S9, S10, S11, S12, S14, S15, S15', S16, S17, S18, S19, S20, S21, S26, S27', and S29) were isolated from three groups (A40, C40, and D40) by ion exchange chromatography on DEAE-cellulose, carboxymethylcellulose, and phosphocellulose and by filtration through Sephadex. The amount of protein obtained varied from 0.1 to 11 mg. Six of the proteins (S5', S10, S11, S18, S19, and S27') had no detectable contamination; the impurities in the others were no greater than 9%. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined.     

Tn10 transposition via a DNA hairpin intermediate

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, a birdlike face, growth retardation, immunodeficiency, lack of secondary sex characteristics in females, and increased incidence of lymphoid cancers. NBS cells display a phenotype similar to that of cells from ataxia-telangiectasia patients, including chromosomal instability, radiation sensitivity, and aberrant cell-cycle-checkpoint control following exposure to ionizing radiation. A recent study reported genetic linkage of NBS to human chromosome 8q21, with strong linkage disequilibrium detected at marker D8S1811 in eastern European NBS families. We collected a geographically diverse group of NBS families and tested them for linkage, using an expanded panel of markers at 8q21. In this article, we report linkage of NBS to 8q21 in 6/7 of these families, with a maximum LOD score of 3.58. Significant linkage disequilibrium was detected for 8/13 markers tested in the 8q21 region, including D8S1811. In order to further localize the gene for NBS, we generated a radiation-hybrid map of markers at 8q21 and constructed haplotypes based on this map. Examination of disease haplotypes segregating in 11 NBS pedigrees revealed recombination events that place the NBS gene between D8S1757 and D8S270. A common founder haplotype was present on 15/18 disease chromosomes from 9/11 NBS families. Inferred (ancestral) recombination events involving this common haplotype suggest that NBS can be localized further, to an interval flanked by markers D8S273 and D8S88.     

Association of unconventional myosin MYO15 mutations with human nonsyndromic deafness DFNB3

DFNB3, a locus for nonsyndromic sensorineural recessive deafness, maps to a 3-centimorgan interval on human chromosome 17p11.2, a region that shows conserved synteny with mouse shaker-2. A human unconventional myosin gene, MYO15, was identified by combining functional and positional cloning approaches in searching for shaker-2 and DFNB3. MYO15 has at least 50 exons spanning 36 kilobases. Sequence analyses of these exons in affected individuals from three unrelated DFNB3 families revealed two missense mutations and one nonsense mutation that cosegregated with congenital recessive deafness.     

In vitro evaluation of 11C-labeled (S)-nicotine, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole, and (R,S)-1-methyl-2-(3-pyridyl)azetidine as nicotinic receptor ligands for positron emission tomography studies

The binding characteristics of the novel 11C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[11C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [11C]MPA in comparison with (S)-[11C]nicotine and [11C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K(D) values) of 2.4 and 560 nM and binding site densities (Bmax values) of 65.5 and 223 fmol/mg of protein for (S)-[11C]nicotine, K(D) values of 0.011 and 2.2 nM and Bmax values of 4.4 and 70.7 fmol/mg of protein for [11C]MPA, and K(D) values of 1.3 and 33.4 nM and Bmax values of 8.8 and 69.2 fmol/mg of protein for [11C]ABT-418. In competing with the 11C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (-)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the 11C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [11C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three 11C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [11C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [11C] MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[11C]nicotine and [11C]ABT-418 raise the level of interest in [11C]MPAfor application in positron emission tomography.