Supporting Appendix 2: Confounding Genes Linked to parkin (Park2) Unless Parkin-deficient mice are compared to control mice on a coisogenic background, there could be systematic and biased differences in genes linked to parkin that could cause phenotypes mistakenly attributed to the parkin mutation. Targeted parkin alleles have been generated using ES cells from 129 mouse strains. When mice carrying these targeted parkin alleles are crossed to the B6 mouse strain, the targeted mutant parkin allele becomes a marker for the 129-derived chromosome; the untargeted parkin allele becomes a marker for the B6 chromosome. On average, genes within a 12-20 cM region of the parkin gene could still be 129 derived in Parkin-deficient mice even after 10-15 backcrosses to B6 mice; the corresponding genes would be B6 derived in control mice (1-3). In earlier backcross generations, this region of difference could be significantly larger (~30 cM after 6 backcrosses). In F2 B6;129 mice, the region of difference could be >60 cM. Moreover, it could take 50 backcrosses to B6 mice to reduce the region of difference between congenic Parkin-deficient and control mice to a ~4 cM region spanning parkin (3). There could be considerable genetic differences between 129 and B6 mice in this relatively small region of the mouse genome (4). Strain differences in genes linked to targeted alleles can produce phenotypes unrelated to the targeted mutation. For example, after 10 backcrosses to create a congenic B6 strain, mice with a targeted deletion of the Il10 gene exhibited reduced exploratory behavior in the open field test (which would be consistent with parkinsonism). This behavioral finding was not due to the Il10 deletion; it was caused by strain differences (129 vs. B6) in a gene linked to the Il10 mutant allele (5). Although these potential confounds apply to many studies using genetically engineered mice, it is of particular concern in Parkin-deficient mice due to the intensity of investigation, the subtle and inconsistent phenotypes, the centromeric location of parkin, and the specific identity of genes linked to parkin. The region of the mouse genome containing parkin, also known as the t complex and located near the MHC locus, is enriched for genes involved in development (6). Within this region there are recombination “cold spots”, from approximately 6.5 to 8 cM for example, where the apparent recombination rates are lower than expected for a random process (7, 8). There are also numerous candidate genes closely linked to parkin that could explain the reported phenotypes in Parkin-deficient mice tested on a B6;129 hybrid genetic background; these genes are very likely 129 derived in Parkin-deficient mice and B6 derived in control mice. In many of these cases, creating Parkin-deficient mice on a congenic B6 background (12 backcrosses) will not fully address the problem because the region of difference, 12-20 cM, still contains confounding genes. Genes of particular concern linked to the parkin locus are identified in the chart below with approximate linkage map distances reported relative to the centromere of Chromosome 17. The parkin gene has been placed at ~6 cM. The chart provides numbered references that illustrate how some of the phenotypes reported in Parkin- deficient mice could be attributed to strain differences (129 vs. B6) in genes closely linked to parkin. References documenting polymorphisms with functional consequences between strains of mice for several of these genes are also provided. Potential confounds of future studies using Parkin-deficient mice on non-coisogenic backgrounds are also identified. Although many examples of potential confounds are illustrated, this FA Perez and RD Palmiter 1 list is not exhaustive; there are additional genes linked to parkin that could further confound interpretation. The genes linked to parkin were identified using the following resources: Mouse Genome Informatics web site (http://www.informatics.jax.org/), UCSC Mouse Genome Bioinformatics web site (http://genome.ucsc.edu/), and NCBI Map Viewer (http://www.ncbi.nlm.nih.gov). Please see the chart on the following page that summarizes the specific confounding genes linked to parkin. References 1. Gerlai, R. (1996) Trends Neurosci 19, 177-81. 2. Gerlai, R. (2001) Behav Brain Res 125, 13-21. 3. Flaherty, L. (1981) in The Mouse in Biomedical Research, eds. Foster, H. L., Small, J. D. & Fox, J. G. (Academic Press, New York), Vol. 1, pp. 215-222. 4. Wade, C. M., Kulbokas, E. J., 3rd, Kirby, A. W., Zody, M. C., Mullikin, J. C., Lander, E. S., Lindblad-Toh, K. & Daly, M. J. (2002) Nature 420, 574-8. 5. Bolivar, V. J., Cook, M. N. & Flaherty, L. (2001) Genome Res 11, 1549-52. 6. Ko, M. S., Threat, T. A., Wang, X., Horton, J. H., Cui, Y., Pryor, E., Paris, J., Wells-Smith, J., Kitchen, J. R., Rowe, L. B., Eppig, J., Satoh, T., Brant, L., Fujiwara, H., Yotsumoto, S. & Nakashima, H. (1998) Hum Mol Genet 7, 1967-78. 7. Schalkwyk, L. C., Weiher, M., Kirby, M., Cusack, B., Himmelbauer, H. & Lehrach, H. (1998) Mamm Genome 9, 807-11. 8. Himmelbauer, H. & Silver, L. M. (1993) Genomics 17, 110-20. FA Perez and RD Palmiter 2 D e cre a Iro A sed M I m e n De n ylo ta cre m a L b Nig cre mp In e o a e id cre a li se ro a t p h rn c se ab ro De De e S in d s a ta De o m tria d S o te cre cre ta se g r u lism m rtl a m a t o sc in De d e- n ficit it r ia lfa P a a in e d och k l ro e d se se e n r m s i e sig ct p in ep cre -i d e r o te ti d d nd ur sp n o s aso b th o a b e n o n ry b ili e sit s o lo u a o m so d f a ty t s Ne ed d com ce n nse o o lin m io y t ce ry p m ria xid ul u u n d a g b e o b ro o b e lo p t l d p NE d n s o m o o h h o a h e ta n r d to co n e e sen ysfu tive e ysfu ur n a cle y we p r g n no n t l su e a m rip o o c o ia a ra cti typ typ sa nct stre t o n to rvi r o y n ct xi ni a ig tu vit ti te ti io p te io g v n h r on st e e on ss e n n ra a ce Gene Symbol cM t e y s s n s t n s l mitochondrial transcription factor B1 Tfb1m, 4 (30) (30) cocaine induced activation 3 QTL Cocia3 4 (18) obesity QTL 4 Obq4 4 (1) (1) NADPH oxidase 3 Nox3 4 (37) phosphodiesterase 10A Pde10a 5 (24,25) (24,25) quaking Qk 6 parkin Park2 6 (57,59,61) (57) (57) (57) (61) (60,61) (57) (58) (59) (59) (57,58) (60) bulb size 4 QTL Bulb4 7 (42) plasminogen Plg 7 (2,3,4,5) (2,23) (2,26,27) (2,38) (2,45) (2,53,54,55) solute carrier family 22, members 1-3 Slc22a1, 2, and 3 7 (46,47,48) acetyl-Coenzyme A acetyltransferases 2 and 3 Acat2 and Acat3 8 (31) (56) superoxide dismutase 2, mitochondrial Sod2 8 (32,33,34,35) (32,33,34,35) (32,33,49) proteasome subunit, beta type 1 Psmb1 8 (43) behavioral response to methamphetamines 10 QTL Brmth10 10 (19) glutamate receptor, metabotropic 4 Grm4 13 (28,29) (39) peroxisome proliferator activator receptor delta Ppard 14 (6,7,8) (6,7,8) body weight, 3 weeks, QTL 3 Wt3q3 14 (9) (9) NADH-ubiquinone oxidoreductase subunit B14.5a Ndufa7 18 (36) proteasome subunit, beta type 9 Psmb9 19 (43,44) proteasome subunit, beta type 8 Psmb8 19 (43,44) tumor necrosis factor Tnf 19 (10,11,12,13) (10,11,12,14,15) (10,11,12,20) (10,11,12,13) (10,11,12,20,50) gamma-aminobutyric acid (GABA-B) receptor, 1 Gabbr1 20 (16) (21) (16) (16) (40,41) ventral midbrain iron content 9 QTL Vmbic9 23 (51) locomotor activity 2 QTL Loco2 23 (17) vascular endothelial growth factor A Vegfa 24 (52) cocaine induced activation 13 QTL Cocia13 25 (22) Chart of confounding genes linked to parkin (Park2). The phenotypes that have been reported (with references identified along the Park2 row) or could be reported in Parkin-deficient mice are identified along the top of the table. Strain differences in genes closely linked to parkin could explain these phenotypes. Numbers reflect references (listed below) that support the role of a parkin-linked gene in a phenotype relevant to the study of Parkin-deficient mice. Distances (cM) are from the centromere of mouse Chromosome 17; parkin is at ~6 cM. Park2 could be considered a candidate gene for some of the QTLs presented; however, studies using Parkin-deficient mice on a B6;129 genetic background are not informative and data using coisogenic Parkin-deficient mice (129S4) are not supportive. FA Perez and RD Palmiter 3 1. Taylor BA, Phillips SJ (1997) Obesity QTLs on mouse chromosomes 2 and 17. Genomics 43:249-257. 2.