Introduction 1995 http://www.goldenretrieverclub.nl 1997 1991 2006 2007 1998 2001 2000 1996 2004 2001 2000 1996 htr1A htr1B htr2A slc6A4 1995 Htr1A 1998 1998 1A 1991 htr1B 2003 2002 HTR1B htr1B 1994 HTR2A 2004 2002 2005 2003 SLC6A4 2003 2002 1996 Slc6A4 2003 2007 Materials and methods Animals, DNA isolation, and phenotyping 1988 2003a 2006 2007 Mutation screening htr1A htr1B htr2A slc6A4 2004 2005 http://www.genetics.bwh.harvard.edu/cgi-bin/pph/polyphen.cgi 1991 1997 1987 Linkage analysis 2003b 2004 2005 1 2004 2005 2004 2005 htr1B htr2A slc6A4 Table 1 Markers used for linkage analysis a b c d d htr1A n  *7370  – htr1B e 157       G/A SNP 246  T/C SNP 955  G/C SNP 1146  n  −68395   htr2A C/T SNP IVS 2-10     n  IVS2 + 1439   slc6A4 C/T SNP 411   G/A SNP IVS9-12  a htr1A htr1B htr2A slc6A4 b c 2001 d htr1A htr2A htr1B slc6A4 e htr1A htr2A htr1B slc6A4 2002 2004 In order to estimate the power of the pedigrees, we calculated the maximum obtainable LOD scores. Affected individuals were assigned haplotypes 2/2 in these calculations; unaffected parents were assigned haplotypes 1/2; and other unaffected individuals were assigned haplotypes 1/1. We assigned haplotypes 0/0 (unknown) to dogs from which we did not have a DNA sample. We assumed that there were four alleles of the hypothetical marker with equal allele frequencies. Association study To test for a more complex genetic effect of variants of the candidate genes, we performed an association study. Fifty aggressive Golden Retrievers were selected from our database. The main selection criteria were high estimated breeding values for human-directed aggression and as little interrelationship among the cases as possible. Non-aggressive dogs were selected for low estimated breeding values for both human- and dog-directed aggression. We avoided an excess of relationship within either the case or the control group. The non-aggressive group was completed with 25 dogs from the random group. A more detailed description of cases and controls is provided in Supplementary Information I. http://www.affymetrix.com htr1A htr1B htr2A slc6A4 2005 P P http://www.pngu.mgh.harvard.edu/~purcell/gpc/ Quantitative genetic analysis 2003 2007 2007 1971 1997 2007 \documentclass[12pt]{minimal}
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$$ y_{ijklmno} = \mu + sex_{i} + age_{j} + htr1a_{k} + htr1b_{l} + htr2a_{m} + slc6a4_{n} + a_{o} + e_{o} $$\end{document} y ijklmno o; μ sex i i age j j j htr1a k  , htr1b l , htr2a m slc6a4 n a o o e o o 4 Results Mutation screening of the coding DNA sequence htr1A htr1B htr2A slc6A4 2004 2005 htr1A htr2A htr1B slc6A4 Linkage analysis htr1A 1 htr1B htr2A slc6A4 htr1B htr2A slc6A4 2 2 htr1A Table 2 a Gene Autosomal dominant  Autosomal recessive htr1A 0.26 −0.30 htr1B −0.72  −2.3 htr2A −1.0  −2.1 slc6A4 0.030 −1.2 Maximum 2.8 5.3 a Association study htr1A htr1B htr2A slc6A4 htr1B slc6A4 P P P htr1A htr1B htr2A slc6A4 3 Table 3 Single nucleotide polymorphisms (SNPs) used for the association study, their allele frequencies in 49 aggressive cases and 49 control dogs, and results of chi-square tests for comparisons of case and control allele frequencies a Chromosomal location b Minor allele frequency in controls Corresponding allele frequency in cases 2 P BICF2P546848 2 51.80 0.29 0.30 0.044 0.83 BICF2P1051894 2 51.96 0.073 0.028 1.6 0.21 BICF2P1398268 2 52.01 0.33 0.33 0.011 0.92 BICF2S23127755 2 52.14 0.23 0.21 0.078 0.78 BICF2P1200391 2 52.22 0.30 0.33 0.21 0.64 BICF2P590055 2 52.35 0.24 0.27 0.19 0.66 BICF2S22939125 2 52.44 0.48 0.53 0.54 0.46 BICF2S23215863 2 52.48 0.30 0.31 0.025 0.87 BICF2P25993 2 52.73 0.49 0.45 0.19 0.66 BICF2S23442706 2 52.76 0.19 0.28 1.8 0.18 Htr1A 2 52.88–52.88 – – – – BICF2P519607 2 53.22 0.27 0.24 0.17 0.68 BICF2P1341930 2 53.62 0.49 0.54 0.52 0.47 BICF2P1159241 12 41.11 0.12 0.13 0.012 0.91 BICF2P555130 12 41.59 0.13 0.21 2.1 0.15 BICF2S23326229 12 41.63 0.24 0.32 1.3 0.25 Htr1B 12 41.65–41.66 – –  – BICF2P670331 12 41.80 0.15 0.23 2.2 0.14 BICF2S23153760 12 41.84 0.039 0.12 3.2 0.073 BICF2P1426522 12 42.29 0.12 0.12 0.013 0.91 BICF2P27571 12 42.34 0.12 0.10 0.21 0.65 BICF2S23444066 12 42.39 0.12 0.11 0.032 0.86 TIGRP2P164447_rs8805986 12 42.51 0.038 0.12 3.6 0.059 BICF2P812153 12 42.57 0.033 0.12 4.8 0.029 BICF2P855402 12 42.62 0.021 0.12 6.9 0.0086 BICF2G630315581 22 6.383 0.10 0.14 0.83 0.36 BICF2G630315746 22 6.611 0.10 0.14 0.45 0.50 BICF2P1168502 22 6.973 0.10 0.11 0.0020 0.96 Htr2A 22 7.395–7.453 – – – – BICF2P164280 22 7.509 0.43 0.54 2.0 0.16 BICF2G630316047 22 7.709 0.44 0.53 1.7 0.19 BICF2S23125159 22 7.866 0.19 0.21 0.047 0.83 BICF2S23661838 22 8.207 0.31 0.36 0.59 0.44 BICF2S22954191 22 8.470 0.23 0.27 0.35 0.55 BICF2P813837 9 46.93 0.39 0.30 1.5 0.22 BICF2S23018060 9 47.06 0.33 0.37 0.22 0.64 BICF2S23551918 9 47.46 0.34 0.38 0.24 0.62 Slc6A4 9 47.55–47.57    – BICF2S23325050 9 47.64 0.11 0.15 0.53 0.47 BICF2S23124809 9 47.79 0.18 0.22 0.38 0.54 BICF2P950384 9 47.88 0.11 0.12 0.032 0.86 BICF2S245135 9 48.02 0.27 0.25 0.074 0.79 BICF2S2347312 9 48.09 0.22 0.28 0.84 0.36 BICF2S23154457 9 48.13 0.31 0.24 1.2 0.28 BICF2S23141984 9 48.26 0.36 0.36 0.0070 0.93 a htr1A htr1B htr2A slc6A4 b http://www.broad.mit.edu/ftp/pub/papers/dog_genome/snps_canfam2/ Canis familiaris http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9615 r 2 htr1A r 2 htr1B htr2A slc6A4 htr1B htr2A slc6A4 htr1A htr1B htr2A slc6A4 3 Quantitative genetic analysis 2007 4 Table 4 htr1A htr1B, htr2A slc6A4 a Number of animals Effect relative to class ‘unknown’ Human-directed aggression Dog-directed aggression htr1A Unknown 44 0.00 0.00 297/297 74 0.028 0.11 297/303 111 0.24 0.054 297/305 15 0.012 0.089 303/303 62 0.18 0.085 303/305 10 −0.11 −0.067 Other 4 −0.046 −0.17 htr1B Unknown 166 0.00 0.00 143-A-G-T-G/143-A-G-T-G 20 −0.73 0.28 143-A-G-T-G /143-C-A-T-G 8 −0.63 −0.19 143-A-G-T-G /139-A-G-C-C 19 0.11 0.19 143-C-A-T-G/143-C-A-T-G 41 −0.17 0.13 143-C-A-T-G /143-A-G-C-C 10 −0.021 0.059 143-C-A-T-G /139-A-G-C-C 30 0.0006 0.32 139-A-G-C-C/139-A-G-C-C 9 0.24 0.11 Other 17 0.15 0.36 htr2A Unknown 114 0.00 0.00 132-C/132-C 40 −0.15 −0.19 132-C/130-C 60 −0.27 −0.023 132-C/132-T 28 −0.22 0.073 132-C/128-C 15 −0.38 −0.43 130-C/130-C 21 −0.33 −0.072 130-C/132-T 22 0.10 −0.19 130-C/128-C 9 −0.52 −0.29 Other 11 0.33 −0.036 slc6A4 Unknown 103 0.00 0.00 C-G/C-G 116 0.025 −0.23 C-G/T-A 94 0.14 −0.23 Other 7 0.079 −0.10 None of the effects was significant a 1 htr1A n n htr1B n n n n n n n htr2A n n n n slc6A4 n n n Discussion We collected behavioral information and DNA samples of 281 dogs over a period of 10 years. Dogs were selected from this collection to evaluate four genes involved in serotonin metabolism by four methods: DNA sequence analysis of the coding region of the genes, genetic linkage analysis, genetic association analysis, and quantitative genetic analysis. The results indicate that it is unlikely that there is a major locus effect of one of the genes on aggression in the Golden Retrievers that we studied. 2005 2004 htr1B htr2A slc6A4 2 htr1B htr2A htr1A slc6A4 htr1A slc6A4 Our linkage analysis does not account for genetic heterogeneity or phenocopies. We have thus only tested for a very strong major locus effect. In reality, the aggressiveness in the Golden Retrievers may be more complex. We therefore used a third study design to investigate the candidate genes: association analysis. For this analysis, we used data from a large-scale genotyping project in 100 Golden Retrievers. Our power calculations demonstrate that this sample size is expected to be sufficient to detect variants that confer a high relative risk for a range of marker allele frequencies. From the total set of 60,073 SNP genotypes, we selected 41 SNPs that flank the candidate genes. We found no association between alleles, genotypes or haplotypes of these SNPs flanking the candidate genes and human-directed aggression of the Golden Retrievers. 2001 2003 1995 1989 1993 2005 Electronic supplementary material Below is the link to the electronic supplementary material.
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