Patrick Charmley

The extent of the human germline T-cell receptor V beta gene segment repertoire

An assessment of the size of the human TCRBV gene segment repertoire based on the identification of TCRBV gene segments in genomic DNA was undertaken. PCR amplification from cloned and uncloned genomic DNA sources, nucleotide sequencing, Southern blot hybridization, and cosmid cloning were used to identify TCRBV gene segments in multiple unrelated individuals. The key advantages to this approach were: (1) TCRBV gene segments which are expressed only at very low levels in cDNA libraries were still detectable, and (2) it was possible to discriminate between alleles at the same locus vs products of different loci. A total of 63 unique TCRBV gene segments were identified and sequenced. Six of these TCRBV gene segments had not been previously described. Thirty-four cosmid clones containing 51 of the 63 identified TCRBV gene segments were isolated and screened for the presence of additional novel TCRBV subfamily members. These results, obtained by a variety of complementary approaches, indicate that the human TCRBV gene segments of which 52 are functional. The availability of the majority of these TCRBV gene segments on cosmid clones should facilitate further investigation of germline TCRBV gene segment polymorphism and putative disease associations.

The germline repertoire of T cell receptor β-chain genes in patients with chronic progressive multiple sclerosis

The T cell receptor (TcR) beta-chain germline gene repertoire of multiple sclerosis (MS) patients was compared to that of 100 normal individuals. No differences in the number of gene segments defined by probes representing 14 different human V beta subfamilies and the constant region genes were found. The distribution of haplotypes defined by restriction fragment length polymorphism (RFLP) alleles detected with V beta 8, V beta 11, and C beta probes in the MS patients was significantly different from that found in normal individuals. Because 84% of the MS patients were DR2+, the findings in these patients were compared to a second group of 43 normals who were DR2+. The distribution of TcR haplotypes in MS patients was also significantly different from that in the DR2+ normals. The data suggest that an MS susceptibility gene(s) may be located in the region of the TcR beta-chain gene complex.

The germline repertoire of T-cell receptor beta-chain genes in patients with multiple sclerosis

The T-cell receptor (TCR) beta-chain gene repertoire of 40 multiple sclerosis (MS) patients was compared to that of 100 normal individuals. V-beta probes that represent 14 different V-beta subfamilies plus a C-beta probe were used to identify 53 separate beta-chain gene segments. No duplication or deletion of any of these 53 gene segments was found in the MS patients. Restriction fragment length polymorphism (RFLP) alleles detected by V-beta 8, V-beta 11 and C-beta probes defined 8 different beta-chain haplotypes. The distribution of these haplotypes in Caucasian MS patients and normal individuals was significantly different (p = 0.012). Comparison of the DR2+ subset of MS patients (n = 32) to a second group of 43 Caucasian DR2+ normal individuals revealed that the distribution of these beta-chain haplotypes was significantly different in these two populations (p = 0.015). These results suggest that an MS susceptibility gene(s) may be located in the region of the TCR/beta-chain gene complex.

Identification of clusters of biallelic polymorphic sequence-tagged sites (pSTSs) that generate highly informative and automatable markers for genetic linkage mapping.

Using a combination of denaturing gradient gel electrophoresis and direct DNA sequencing, we have found that multiple (4 to 7) biallelic sequence polymorphisms can be located within short DNA segments, 300 to 2400 bp. Here, we report on the identification of three clusters of DNA polymorphisms, one in each of the constant regions of the human T cell receptor alpha and beta gene complexes on human chromosomes 14 and 7, respectively, and a third among the human t-RNA genes on human chromosome 14. The frequency of these polymorphisms and the extent of linkage disequilibrium between individual polymorphisms have been determined using a semiautomated DNA typing system combining DNA target amplification by the polymerase chain reaction with the analysis of internal sequence polymorphisms by a colorimetric oligonucleotide ligation assay. We have found that individual biallelic polymorphisms in each cluster are often in partial linkage disequilibrium with one another. This partial linkage disequilibrium permits the combined use of three to four markers in a cluster to generate a haplotype with high levels of heterozygosity, 71 to 88%. Therefore, clusters of physically linked biallelic polymorphisms provide an automatable and highly informative type of genetic marker for general linkage analysis as well as an attractive alternative marker system for fine-point mapping of disease-causing genes and phenotypic traits relative to their framework locations in the genome.

RNAi-based Therapeutics Targeting Survivin and PLK1 for Treatment of Bladder Cancer

Harnessing RNA interference (RNAi) to silence aberrant gene expression is an emerging approach in cancer therapy. Selective inhibition of an overexpressed gene via RNAi requires a highly efficacious, target-specific short interfering RNA (siRNA) and a safe and efficient delivery system. We have developed siRNA constructs (UsiRNA) that contain unlocked nucleobase analogs (UNA) targeting survivin and polo-like kinase-1 (PLK1) genes. UsiRNAs were encapsulated into dialkylated amino acid-based liposomes (DiLA(2)) containing a nor-arginine head group, cholesteryl hemisuccinate (CHEMS), cholesterol and 1, 2-dimyristoyl-phosphatidylethanolamine-polyethyleneglycol 2000 (DMPE-PEG2000). In an orthotopic bladder cancer mouse model, intravesical treatment with survivin or PLK1 UsiRNA in DiLA(2) liposomes at 1.0 and 0.5 mg/kg resulted in 90% and 70% inhibition of survivin or PLK1 mRNA, respectively. This correlated with a dose-dependent decrease in tumor volumes which was sustained over a 3-week period. Silencing of survivin and PLK1 mRNA was confirmed to be RNA-induced silencing complex mediated as specific cleavage products were detected in bladder tumors over the duration of the study. This report suggests that intravesical instillation of survivin or PLK1 UsiRNA can serve as a potential therapeutic modality for treatment of bladder cancer.

An Amino Acid-based Amphoteric Liposomal Delivery System for Systemic Administration of siRNA

We demonstrate a systematic and rational approach to create a library of natural and modified, dialkylated amino acids based upon arginine for development of an efficient small interfering RNA (siRNA) delivery system. These amino acids, designated DiLA₂ compounds, in conjunction with other components, demonstrate unique properties for assembly into monodisperse, 100-nm small liposomal particles containing siRNA. We show that DiLA₂-based liposomes undergo a pH-dependent phase transition to an inverted hexagonal phase facilitating efficient siRNA release from endosomes to the cytosol. Using an arginine-based DiLA₂, cationic liposomes were prepared that provide high in vivo siRNA delivery efficiency and are well-tolerated in both cell and animal models. DiLA₂-based liposomes demonstrate a linear dose-response with an ED₅₀ of 0.1 mg/kg against liver-specific target genes in BALB/c mice.

Further localization of a multiple sclerosis susceptibility gene on chromosome 7q using a new T cell receptor beta-chain DNA polymorphism

Multiple sclerosis (MS) has been associated with particular HLA haplotypes and has recently been reported to also be associated with the T cell receptor (TCR) beta-chain complex. We have tried to determine the source of the TCR-beta/MS association by exploiting the pattern of linkage disequilibrium within the TCR-beta complex. We describe a new DNA polymorphism with the TCR variable region gene segment V beta 15 which appears to localize between the constant region and V beta 11. When the distribution of V beta 11-V beta 15 haplotypes in MS patients was compared to healthy controls, the strength of the V beta 11-V beta 15 MS association (p = 0.107) was much less than the MS association with the adjacent V beta 8-V beta 11 haplotype (p = 0.0010). On the basis we exclude an MS susceptibility gene telomeric to V beta 11. The reported MS association with the TCR-beta gene complex therefore does not appear to be due to genes within the diversity, joining, or constant region but more likely involves a specific gene(s) within the variable region.

PCR-BASED GENOTYPING AND HAPLOTYPE ANALYSIS OF HUMAN TCRBV GENE SEGMENT POLYMORPHISMS

There are at least 63 tandemly arranged human T-cell receptor (Tcr) β-chain variable region (BV) gene segments, which have presumably arisen by repeated gene duplication events. The 5′-most half of the TCRBV gene loci is particularly complex in organization due to the presence of multiple interspersed members of the largest BV subfamilies, BV5, BV6, and BV13. Polymorphism and linkage relationships among these genes has been poorly characterized in part due to the high similarity of these duplicands. Germline DNA polymorphisms were specifically examined in the exons and introns of these and other BV gene segments distributed across 240 kilobases (kb) in this 5′-most region. Polymerase chain reaction restriction enzyme-based assays were used to genotype ten point mutations in seven of the BV gene segments. Eight of these polymorphisms altered an amino acid of the BV gene segment. In addition, length polymorphisms due to simple sequence repeats were noted in the introns of six BV6 subfamily members. Approximately 250 unrelated haplotypes were constructed by segregation analyses of fifteen of these TCRBV polymorphisms. Linkage disequilibrium analyses indicated that haplotypic relationships are not detectable over a distance of more than 55 kb in this genomic region. These TCRBV polymorphisms, and the haplotypic analysis, provide important resources and guidance for future attempts to associate Tcr germline DNA differences in the human population with immune response differences, such as might occur in some autoimmune diseases.

Polymorphism and phylogeny of dinucleotide repeats in human T-cell receptor Vb6 genes

The Vb6 subfamily is the largest reported subfamily of human T-cell receptor (Tcr) genes with as many as 14 possible members based on variation in reported DNA sequences. A study of the genomic organization of four distinct Vb6 genes indicated that they contained within their introns theuniterrupted dinucleotide repeat (GT)n, with n>8. DNA amplification primers and conditions were determined which amplified the intron of these four different Vb6 gene segments. All four Vb6 genes tested showed length polymorphism when examined in a group of unrelated individuals. Careful sizing and DNA sequencing showed that the alleles of each gene differed in size by multiples of two base pairs (bp), due to different repeat numbers of the dinucleotide (GT)n. These four microsatellite polymorphisms had from three to ten alleles, and individual heterozygosities of 26% to 83%. The large number of alleles and the high heterozygosity make these polymerase chain reaction (PCR)-based polymorphisms very attractive genetic markers for segregation studies which postulate the presence of autoimmune susceptibility genes within the Tcrb region. Vb6 hybridization to genomic DNA confirmed the relatively large size of the Vb6 subfamily in several hominoid species. Nucleotide sequencing of an intron of the Vb6 genes from other primates revealed the presence of dinucleotide repeats similar to those found in human Vb6 genes. Thus, the (GT)n microsatellite was not only present in the Vb6 intron before Vb6 gene duplication, but was present before speciation of the hominoids.

Genetic linkage of the human apolipoprotein AI-CIII-AIV gene cluster and the neural cell adhesion molecule (NCAM) gene

The genes encoding apolipoproteins AI, CIII, and AIV, three plasma proteins involved in lipid metabolism, are clustered within a 15-kb DNA segment (apoAI-CIII-AIV gene cluster) located on human chromosome 11 at band q23. The gene encoding the neural cell adhesion molecule (NCAM), a cell surface glycoprotein involved in cell-cell recognition during morphogenesis, is also located on chromosome 11, band q23. In this report, 12 previously described restriction fragment length polymorphisms (RFLPs) in the apoAI-CIII-AIV gene cluster were tested for cosegregation with a newly identified BamHI RFLP in the NCAM gene using 13 families. The results show that the apoAI-CIII-AIV gene cluster and the NCAM gene loci are linked with a maximum lod score of 15.9 at a recombination fraction of 0.028. In addition, an approach for the most efficient use of the apoAI-CIII-AIV gene cluster polymorphisms, based on the evaluation of their individual and cumulative heterozygosities, is presented.