Patricia Taillon-Miller

Juxtaposed regions of extensive and minimal linkage disequilibrium in human Xq25 and Xq28

Linkage disequilibrium (LD), or the non-random association of alleles, is poorly understood in the human genome. Population genetic theory suggests that LD is determined by the age of the markers, population history, recombination rate, selection and genetic drift. Despite the uncertainties in determining the relative contributions of these factors, some groups have argued that LD is a simple function of distance between markers. Disease-gene mapping studies and a simulation study gave differing predictions on the degree of LD in isolated and general populations. In view of the discrepancies between theory and experimental observations, we constructed a high-density SNP map of the Xq25–Xq28 region and analysed the male genotypes and haplotypes across this region for LD in three populations. The populations included an outbred European sample (CEPH males) and isolated population samples from Finland and Sardinia. We found two extended regions of strong LD bracketed by regions with no evidence for LD in all three samples. Haplotype analysis showed a paucity of haplotypes in regions of strong LD. Our results suggest that, in this region of the X chromosome, LD is not a monotonic function of the distance between markers, but is more a property of the particular location in the human genome.

The optimal measure of allelic association

Allelic association between pairs of loci is derived in terms of the association probability ρ as a function of recombination θ, effective population size N, linear systematic pressure v, and time t, predicting both ρrt, the decrease of association from founders and ρct, the increase by genetic drift, with ρt = ρrt + ρct. These results conform to the Malecot equation, with time replaced by distance on the genetic map, or on the physical map if recombination in the region is uniform. Earlier evidence suggested that ρ is less sensitive to variations in marker allele frequencies than alternative metrics for which there is no probability theory. This robustness is confirmed for six alternatives in eight samples. In none of these 48 tests was the residual variance as small as for ρ. Overall, efficiency was less than 80% for all alternatives, and less than 30% for two of them. Efficiency of alternatives did not increase when information was estimated simultaneously. The swept radius within which substantial values of ρ are conserved lies between 385 and 893 kb, but deviation of parameters between measures is enormously significant. The large effort now being devoted to allelic association has little value unless the ρ metric with the strongest theoretical basis and least sensitivity to marker allele frequencies is used for mapping of marker association and localization of disease loci.

Yeast artificial chromosome cloning of a two-megabase-size contig within chromosomal band 18q21 establishes physical linkage between BCL2 and plasminogen activator inhibitor type-2

The construction of large-scale physical maps requires efficient approaches to generate new probes and link informative markers. The mapping of a human chromosomal segment was initiated by using the 18q21.3 probes, plasminogen activator inhibitor type-2 (PLANH2) and BCL2, to screen a yeast artificial chromosome (YAC) library. An inverse polymerase chain reaction technique rescued genomic ends of the YAC inserts. These new probes were used in a chromosomal walking strategy which established that the PLANH2 gene was 600 kb telomeric and in the opposite transcriptional orientation to that of BCL2. Overall, 16 YACs with a mean size of approximately 300 kb were analyzed using rare-cutting restriction endonucleases and 10 end-rescued probes. A contiguous map within 18q21.3 that spans approximately 2 Mb was assembled. This establishes the feasibility of using YACs in the efficient cloning and physical surveying of expanses of the human genome lacking closely spaced, genetic landmarks.

The Immunogenetics of Smallpox Vaccination

We hypothesized that individuals who develop fever after smallpox vaccination have genetically determined differences in their immune responses to vaccinia virus. We looked for an association between the development of fever and single-nucleotide polymorphisms (SNPs) in 19 candidate genes in 346 individuals previously assessed for clinical responses to smallpox vaccination. Fever after smallpox vaccination is associated with specific haplotypes in the interleukin (IL)-1 gene complex and in the IL18 gene. A haplotype in the IL4 gene was highly significant for reduced susceptibility to the development of fever after vaccination among vaccinia-naive individuals. Our results indicate that certain haplotypes in the IL-1 gene complex and in IL18 and IL4 predict an altered likelihood of the development of fever after smallpox vaccination. Our findings also raise the possibility that these same haplotypes may identify individuals at risk for the development of fever after receipt of other live virus vaccines, providing information that could be useful in anticipating and preventing more-serious adverse events.

A Yeast Artificial Chromosome Contig Encompassing the Type 1 Neurofibromatosis Gene

The yeast artificial chromosome (YAC) system (Burke et al., 1987, Science 236: 806-812) allows the direct cloning of large regions of the genome. A YAC contig map of approximately 700 kb encompassing the region surrounding the type 1 neurofibromatosis (NF1) locus on 17q11.2 has been constructed. A single YAC containing the entire NF1 locus has been constructed by homologous recombination in yeast. In the process of contig construction a novel method of YAC end rescue has been developed by YAC circularization in yeast and plasmid rescue in bacteria. YACs containing homology to the NF1 region but mapping to another chromosome have also been discovered. Sequences of portions of the homologous locus indicate that this other locus is a nonprocessed pseudogene.

Physical mapping of yeast artificial chromosomes containing sequences from the human β-globin gene region

The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human beta-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult beta-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire beta-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the beta-globin gene cluster.

The Tetratricopeptide repeat domain 7 gene is mutated in flaky skin mice: a model for psoriasis, autoimmunity, and anemia.

The flaky skin (fsn) mutation in mice causes pleiotropic abnormalities including psoriasiform dermatitis, anemia, hyper-IgE, and anti-dsDNA autoantibodies resembling those detected in systemic lupus erythematosus. The fsn mutation was mapped to an interval of 3.9 kb on chromosome 17 between D17Mit130 and D17Mit162. Resequencing of known and predicted exons and regulatory sequences from this region in fsn/fsn and wild-type mice indicated that the mutation is due to the insertion of an endogenous retrovirus (early transposon class) into intron 14 of the Tetratricopeptide repeat (TPR) domain 7 (Ttc7) gene. The insertion leads to reduced levels of wild-type Ttc7 transcripts in fsn mice and the insertion of an additional exon derived from the retrovirus into the majority of Ttc7 mRNAs. This disrupts one of the TPRs within TTC7 and may affect its interaction with an as-yet unidentified protein partner. The Ttc7 is expressed in multiple types of tissue including skin, kidney, spleen, and thymus, but is most abundant in germinal center B cells and hematopoietic stem cells, suggesting an important role in the development of immune system cells. Its role in immunologic and hematologic disorders should be either investigated.

THE HUMAN FACTOR H-RELATED GENE 2 (FHR2): STRUCTURE AND LINKAGE TO THE COAGULATION FACTOR XIIIB GENE

The human factor H-related gene 2 (FHR2) encodes a serum protein structurally and immunologically related to complement factor H. We describe the isolation and genomic organization of the human FHR2 gene from a yeast artificial chromosome library. The FHR2 gene is organized in five exosn and span about 7 kilobases (kb) of human genomic DNA. A comparison with the corresponding cDNA sequence (clone DDESK59) shows that the analyzed FHR2 gene has a deleted region within exon 4. A new splice acceptor site created in the truncated exon indicates that the analyzed gene could be translated to a truncated protein. Further, we demonstrate that the genes for FHR2 and β subunit of coagulation factor XIII are located in the same 165 kb YAC DNA. Thus, the three structurally related genes FXIIIb, FHR2, and factor H are linked on human chromosome 1 in the regulators of complement activiation (RCA) gene cluster. The physical linkage of the FHR2 and the factor H genes provides additional evidence for a close relatedness of complement factor H and the factor H-related proteins. The linkage and the almost exclusive organization in short consensus repeat-containing domains indicates a close evolutionary relationship of the FXIIIb, FHR2, and factor H genes.

Isolation and characterization of a yeast artificial chromosome (YAC) contig around the human steroid sulfatase gene

The region surrounding the steroid sulfatase (STS) locus on Xp22.3 is of particular interest since it represents a deletion hot spot, shares homology with the proximal long arm of the Y chromosome (Yq11.2), and contains genes for several well-described X-linked disorders. Here we describe yeast artificial chromosomes (YACs) covering 450 kb around the STS gene. Eight YAC clones were isolated from a human YAC library. Their STS exon content was determined and the overlap of the clones characterized. Two of the YAC clones were found to contain the entire STS gene. The most proximal and the most distal ends of the YAC contig were cloned but neither of them crossed the breakpoints in any of the previously described patients with entire STS gene deletions. This is consistent with deletions larger than 500 kb in all these patients. One of the YAC clones was found to contain sequences from the STS pseudogene on Yq11.2. Two anonymous DNA sequences, GMGXY19 and GMGXY3, previously mapped in the vicinity of the STS locus, were found within the YAC contig and their assignment with respect to the STS locus was thus possible. This contig is useful for the overlap cloning of the Xp22.3 region and for reverse genetic strategies for the isolation of disease genes in the region. Furthermore, it may provide insight into the molecular mechanisms of deletion and translocation events on Xp22.3 and in the evolution of sex chromosomes.

Molecular linkage of the HLA-DR, HLA-DQ, and HLA-DO genes in yeast artificial chromosomes

Eight major histocompatibility complex (MHC) class II loci and the newly defined Y3/Ring 4 locus were isolated in overlapping yeast artificial chromosome (YAC) clones defining a 420-kb segment of human chromosome 6p21.3. YAC B1D12 spanning 320 kb contained seven of these loci from HLA-DRA to HLA-DQB2. A 330-kb YAC, A148A7, spanned from the HLA-DQA1 locus through the Y3/Ring 4 locus and extended at least 130 kb centromeric of YAC B1D12. Southern blotting demonstrated that YAC B1D12 derived from the HLA-DR3 haplotype and that YAC A148A7 derived from the HLA-DR7 haplotype of the heterozygous library donor. A third 150-kb YAC, A95C5, lay within this contig and contained only the HLA-DRA locus. A fourth 300-kb YAC, A76F11, was isolated by chromosome walking from the telomeric end of YAC B1D12. Probes isolated from the ends of the YAC genomic inserts have been used to confirm overlaps between the clones. These analyses demonstrated that the centromeric end of YAC A76F11 used the same genomic EcoRI cloning site as the telomeric end of YAC A95C5. YAC B1D12 used an EcoRI site only 2.1 kb telomeric of the aforementioned EcoRI site. These data suggest that certain EcoRI sites are used preferentially during construction of the library. These YACs complete the linkage of the DR and DQ subregions of the HLA complex in cloned DNA and provide the substrate for precise analysis of this portion of the class II region.