Genome sequence based, comparative analysis of the fluorescent amplified fragment length polymorphisms (FAFLP) of tubercle bacilli from seals provides molecular evidence for a new species within the Mycobacterium tuberculosis complex.

Tuberculosis in seals is caused by a member of the Mycobacterium tuberculosis complex referred to as the 'seal bacillus'. Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was applied to isolates from four Australian and six Argentinean seals and compared with FAFLP pattern for standard strains belonging to the M. tuberculosis complex. The FAFLP profiles derived from EcoRI/MseI restricted fragments of blind coded DNA samples differentiated the seal bacillus from other members of the M. tuberculosis complex. According to the phylogenetic analysis performed using FAFLP data, seal bacilli appear to have diverged significantly from other members of the M. tuberculosis complex. We describe the suitability of a panel of 19 highly polymorphic markers for rapid identification and comparative genomic analyses of the seal bacillus strains. It is likely that these bacilli got separated from the M. tuberculosis lineage as a result of different insertion deletion events occurring on a genome wide scale. Our analysis reveals that the seal bacillus and M. bovis are genetically related and therefore, might have originated from a common ancestor. Our data additionally support the hypothesis that seal bacillus occupies a unique taxonomic position within the M. tuberculosis complex.     

Identification of a gene disrupted by a microdeletion in a patient with X-linked retinitis pigmentosa (XLRP)

The gene for the most frequent from of X-linked retinitis pigmentosa (XLRP), RP3, has been assigned by genetic and physical mapping to a segment of less than 1000 kbp, which is flanked by the marker DXS1110 and the ornithine transcarbamylase (OTC) gene. In search of microdeletions, we have screened the DNA of 30 unrelated patients with XLRP by employing a representative set of YAC-derived DNA fragments that were generated by restriction enzyme digestion and PCR amplification. In one of these patients, a 6.4 kbp microdeletion was detected which was not present in the DNA of 444 male controls. A cosmid contig spanning the deletion was constructed and used to isolate cDNAs from retina-specific libraries. Exons corresponding to these expressed sequences as well as other putative exons were identified by sequencing more than 30 kbp of the critical region. So far, no point mutations in these putative exon sequences have been identified.      

Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides

We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membrane-bound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian population. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptide-binding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.     

Linkage of chromosome 13q32 to schizophrenia in a large veterans affairs cooperative study sample

Several prior reports have suggested that chromosomal region 13q32 may harbor a schizophrenia susceptibility gene. In an attempt to replicate this finding, we assessed linkage between chromosome 13 markers and schizophrenia in 166 families, each with two or more affected members. The families, assembled from multiple centers by the Department of Veterans Affairs Cooperative Studies Program, included 392 sampled affected subjects and 216 affected sib pairs. By DSM-III-R criteria, 360 subjects (91.8%) had a diagnosis of schizophrenia and 32 (8.2%) were classified as schizoaffective disorder, depressed. The families had mixed ethnic backgrounds. The majority were northern European-American families (n = 62, 37%), but a substantial proportion were African-American kindreds (n = 60, 36%). Chromosome 13 markers, spaced at intervals of approximately 10 cM over the entire chromosome and 2-5 cM for the 13q32 region were genotyped and the data analyzed using semi-parametric affected only linkage analysis. For the combined sample (with race broadly defined and schizophrenia narrowly defined) the maximum LOD score was 1.43 (Z-score of 2.57; P = 0.01) at 79.0 cM between markers D13S1241 (76.3 cM) and D13S159 (79.5 cM). Both ethnic groups showed a peak in this region. The peak is within 3 cM of the peak reported by Brzustowicz et al. [1999: Am J Hum Genet 65:1096-1103].     

HLA-DRB1 molecules and antigenic experience shape the repertoire of CD4 T cells

Forces influencing the composition of the mature TCR repertoire have been well studied in the mouse. In particular, the contribution of MHC molecules in negative and positive selection events of T lymphocytes has been established. To understand whether the allelic polymorphism of HLA-DRB1 molecules can shape the human TCR repertoire, we compared the usage of TCR Vβ segments in two cohorts of unrelated individuals who were selected for the expression of HLA-DRB1 alleles. To investigate the potential role of antigenic experience in shaping the TCR repertoire, we compared the usage of Vβ gene elements in CD45RO⁻ CD4⁺ (naive) T cells versus CD45RO⁺ CD4⁺ (memory) T cells. A correlation between Vβ gene segment usage and HLA-DRB1 alleles could be demonstrated for the repertoire of the naive CD4⁺ T cells, suggesting a shaping force of the HLDRB1 allele on the peripheral TCR repertoire. While the HLA-DRB1 imposed profile in Vβ distribution was maintained in CD45RO⁺ CD4⁺ T cells, it was less pronounced, indicating that antigenic experience modulates the functional TCR repertoire.