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Show 166 GENETIC ANALYSIS OF LYME DISEASE Heydon Kaddas (Janis Weis) Department of Pathology University of Utah RESEARCH POSTERS ON THE HILL SPRING 2012 Genetics of Lyme Arthritis Heydon Kaddas and Dr. Janis Weis Department of Pathology Examining single nucleotide polymorphisms (SNP's) between the C3H/HeN and C57/BL6 strains in the transgressed region has identified roughly 16 genes of interest, indicated by the colored lines. All of these genes are known to play a role in the immune system. Particularly interesting are the three Nlrp genes, as they have been implicated in other inflammatory diseases, as well as the Csf2 and Il3 genes, which interact with one another and have been shown to resolve inflammation. Depicted are the results from a total genome scan of an initial infection experiment. Arthritis was assessed after infection of C57/BL6 mice, C3H/HeN mice, the offspring of a cross between the two strains (F1 generation) and populations of the F1 generation crossed back to each parental (C57/BL6 or C3H/HeN) strain. Each gray and red image is a chromosome , as indicated below by the abbreviation and associated number. The black lines next to each chromosome represents a region on that chromosome that were identified as playing a role in the development of Lyme Arthritis. The names of each region are indicated below the chromosome number. Lyme disease is a tick-transmitted infection caused by the spirochete Borrelia burgdorferi. Some patients exhibit severe tendonitis from inflammation while others display few to no symptoms. Symptom variance is also seen in mouse strains CH3/HeN and C57BL/6 where arthritis severity is respectively severe and moderate. Heydon Kaddas Dr. Janis Weis C57/BL6 F1(B6.C3-Bbaa4 x B6) F1 x BL6 1. Generate recombinants on the BL6 background by breeding C57/BL6 mice to C3H/HeN mice. 1. From the offspring of the above cross, identify interval-specific recombinants: B6.C3H-Bbaa4. 3. Further disrupt the interval by breeding the B6.C3H-Bbaa4 mice to the C57/BL6 mice. 4. Identify mice with smaller regions of C3H/HeN DNA. Fix the interval, infect and assess arthritis. 5. Continue breeding to narrow interval to 1-2 cM, or 0.5Mbp B6.C3-Bbaa4 B B B B B B B B B B 1 2 3 4 5 6 3Mbp 110Mbp 58Mbp C C C C C C C C C C C C B B B B B B B B B B B B Sex Mb 6 17 26 35 45 54 62 63 64 68 70 71 72 74 75 76 81 82 83 89 99 101 109 F2 B B B B B B B B C C C C C C C C C C C C C C B M3 B B B B B B B B C C C C C C C C C C C C C C B F3 B B B B B B C C C C C C C C C C C C C C C C B M1 B B B B B B C C C C C C C C C C C C C C C C B M29 B B B B B B B B B B B B H H H H H H H H H H B F45 B B B B B B B B B B B B H H H H H H H H H H B M7 C C C C C C C C H B B B B B B B B B B B B B B F7 C C C C C C C C C B B B B B B B B B B B B B B F2 C C C C C C B B B B B B B B B B B B B B B B B M4 C C C C C C B B B B B B B B B B B B B B B B B F66 H H H H H H H H H H H H B B B B B B B B B B B M3 H H H H C C C C C C C C H H H B B B B B B B B 9 Mbp 26 36 40 44 54 63 70 83 84 94 101 113 d11m226 d11m51 d11m110 d11m235 d11m350 d11m120 d11m35 d11m228 d11m359 d11m12 Txd Region QTL LOD Score -Trait 9 Chr. 11 Nlrp3 Glp2r Nlrp1A Itgae Nlrp1B Mmp28 Csf2 Expi Il3 Ap1gbp1 Igtp Trim25 Lrrc48 Itga3 Trim16 Spnb2 Chr 5 Chr4 Bbaa2Bbaa3 Chr 1 Bbaa 1 Bbaa 12 Chr 11 Bbaa4 Chr 12 Bbaa 6 Six Lyme Arthritis associated regions (labeled Bbaa) have been previously identified on five chromosomes. This illustrates the genotype of breeder pairs on chromosome 11. The numbers at the top represent Single Nucleotide Polymorphism (SNP) markers spanning the transgressed region of C3H/HeN DNA on chromosome 11. Numbers highlighted in blue indicate the Bbaa4 region identified in the QTL analysis. A red C indicates the animal is homozygous for the C3H/HeN allele at that position. White B's indicate that an animal is homozygous for the C57/BL6 allele. A yellow H indicates that the animal is heterozygous, containing both the C3H/HeN and C57/BL6 allele, at the position. The blue label on the left hand side distinguishes each of the breeder pairs. Before infection with B. burgdorferi all strains must be homozygous for the C3H/HeN allele in current heterozygous positions. This illustrates the desired chromosome 11 for each breeding population 1-6. Red regions with a black C indicate that all base pairs in the region are identical to the C3H/HeN parent. Gray regions with a white B indicate that all base pairs in the region are identical to the C57/BL6 parent. In order to identify possible genes in a red region contributing to Lyme Arthritis, the mice must have 2 identical chromosomes as shown. The above text describes the plan for the overall experiment to investigate the contribution of the Bbaa4 region on chromosome 11 to arthritis severity. On the left hand side, the breeding process is illustrated. Brown triangles indicate C3H/HeN DNA present on chromosome 11. The B6.C3-Bbaa4 mice have almost the entire chromosome with C3H/HeN DNA present. As shown in each subsequent generation, continued breeding to C57/BL6 background decreases the region on chromosome 11 that contains C3H/ HeN DNA. Mice containing the most similar regions of C3H/HeN DNA are then selected from the final generation shown and bred together to obtain the populations that will be infected and analyzed. C57BL/6 (B6) Mild Arthritis C3H/HeN Severe Arthritis Lyme disease is transmitted through tick bites from Ixodes scapularis, more commonly called deer ticks. About 30 % of patients infected with B. Burgdorferi will develop the chronic inflammatory symptoms known as Lyme Arthritis. Lyme disease is caused by the spirochete Borrelia Burgdorferi. Like humans, different strains of mice develop varying degrees of severity of Lyme arthritis. Ixodes Scapularis at the nymphal (top), larval (middle) and adult stages of their life cycle The knee joints of a patient presenting development of Lyme Arthritis in the left most knee. Imaging of many Borrelia Burgdorferi Introduction to Lyme Arthritis Identification of Quantitative Trait Loci (QTL) Development of Interval Specific Recombinant Congenic Lines (ISRCL) B6.C3H- Bbaa4 ISCRL Chromosome Illustration Current breeding B6.C3H- Bbaa4 ISCRL Populations Genes of Interest Lyme disease is a tick-transmitted infection caused by the spirochete Borrelia burgdorferi. Some patients exhibit severe tendonitis from in ammation while others display few to no symptoms. Symptom variance is also seen in mouse strains CH3/HeN and C57BL/6 where arthritis severity is respectively severe and moder ate. Intercross populations of these strains sug gest multiple regions of genetic linkage (QTL or Quantitative Trait Loci) control the observed di erences. To better understand this disease, the regions of linkage must be investigated. Regions of identi ed linkage from C3H/HeN mice have been crossed on to the C57BL/6 background. This has established multiple strains of congenic mice, each homozygous for the suggested region. One of these strains, B6.C3-Bbaa4, has a transferred region on chromosome 11. Examining single nucleotide poly-morphisms (SNP's) between strains in the region has identi ed roughly 10 genes of interest; Nlrp3, Nlrp1a, Nlrp1b, Csf2, Il3, Igtp, Lcrr48, Trim 16, Glp2r and Itage. All of these genes are known to play a role in the immune system. Particularly interesting are the three Nlrp genes, as they have been implicated in other in ammatory diseases, as well as the Csf2 and Il3 genes, which interact with one another and have been shown to resolve in ammation. To determine if these genes, or other closely linked genes, are impacting arthritis severity, the QTL interval needs to be broken into smaller regions of C3H/HeN homozygosity. Breeding within the B6.C3-Bbaa4 strain has already yielded signi cant progress towards this goal. We are generating six recombinant congenic lines spanning the QTL region on chromosome 11. The in uence of C3H alleles in these regions will be determined by infecting approximately 10 mice from each strain with B. burgdorferi. The severity of arthritis in these strains should give us insight into the e ect of narrowed regions and allow identi cation of possible candidate genes a ecting arthritis severity. |