Supplementary Information A duplication of FGF3, FGF4, FGF19 and ORAOV1 causes the hair ridge and predisposes to dermoid sinus in Ridgeback dogs Nicolette H. C. Salmon Hillbertz 1, Magnus Isaksson 2, Elinor K. Karlsson 3,4, Eva Hellmén 2,5, Gerli Rosengren Pielberg 6, Peter Savolainen 7, Claire M. Wade 3,8, Henrik von Euler 9, Ulla Gustafson 1, Åke Hedhammar 9, Mats Nilsson 2, Kerstin Lindblad-Toh 3, Leif Andersson 1,6 & Göran Andersson 1 1 Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences (SLU), Biomedical centre, Box 597, SE-751 24 Uppsala, Sweden. 2 Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden. 3 Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA. 4 Bioinformatics Program, Boston University, 44 Cummington Street, Boston, Massachusetts 02215, USA. 5 Department of Anatomy, Physiology and Biochemistry, SLU, Box 7011, SE-750 07 Uppsala, Sweden. 6 Department of Medical Biochemistry and Microbiology, Uppsala University, Box 597, SE-751 24 Uppsala, Sweden. 7 School of Biotechnology, KTH, Royal Institute of Technology, AlbaNova University Center, SE-106 91 Stockholm, Sweden. 8 Center for Human Genetic Research, Massachusetts General Hospital, Boston MA 02114 USA. 9 Department of Clinical Sciences, SLU, Box 7054, SE-750 07 Uppsala, Sweden. 1
Supplementary Methods Animals. The Rhodesian Ridgebacks included in this study were derived from Scandinavian dog populations and the Thai Ridgebacks were derived from European and Thai dog populations. The collection of field material (whole blood and tissues) was performed in accordance with the animal ethics regulations of the Swedish National Board for Laboratory Animals and the Swedish Board of Agriculture. DS-affected Rhodesian Ridgeback puppies were euthanized and autopsied at the Swedish University of Agricultural Sciences. Histopathological confirmation was performed to ensure correct clinical diagnosis for all Rhodesian Ridgeback dogs while information regarding DS in the Thai Ridgebacks was obtained from owners. Genomic DNA from whole blood was extracted by standard methods. Total RNA from tissues was extracted by standard TRIzol protocol. Histology. The skin was cut strictly in the median plane from the ridge and from the corresponding region in the ridgeless dog. Tissues from the skin and dermoid sinus were fixated in 4% buffered formalin and embedded in paraffin according to standard procedures. Sections were cut at 5µm and stained with hematoxylin and eosin. Digital light micrographs were taken using a Nikon Eclipse, E1000 microscope. Multiplex Ligation dependent Genome Amplification (MLGA). MLGA was essentially performed according to Isaksson et al. 1, with the following modifications: 12-200 ng DNA was restriction digested for 1 h at 25 C using 4 U of restriction enzyme CviA II (New England Biolabs) in 10 µl NEB4 buffer (New England Biolabs) supplemented with 0.1 µg/µl BSA. The restriction enzyme was heat inactivated at 65 C for 20 min. Circularization of restriction fragments, exonuclease I treatment, PCR amplification, and fluorescent readout using Agilent Bioanalyzer 2100, were all performed as described 1. All measurements were done in duplicate or triplicate. 2
Breakpoint analysis. Breakpoint-specific PCR was performed by adding 4 µl (~ 15 ng) DNA to 21 µl of a PCR-mix containing 1.2 x PCR buffer (buffer 1 from Roche kit, cat. no. 11 681 834 001), 0.42 mm dntp, 0.36 µm each of P1 forward and reverse primers (Supplementary Table 1), and 1.9 U Platinum Taq polymerase (Invitrogen). PCR amplification was performed according to standard protocols. Sequencing was performed on a MegaBACE1000 and analysis was conducted with Sequencher software v 3.1.1 (GeneCodes). The sequences were compared to the available CanFam2.0 genome sequence (http://genome.ucsc.edu/) using BLAT. Sequence analysis of mitochondrial DNA. 582 bp of the control region (D-loop) was analysed. Buccal epithelial cell samples were collected using Whatman FTA-indicating cards according to the manufacturer s specifications. PCR and DNA sequence analysis were performed as described 2. Eleven Rhodesian Ridgebacks and eight Thai Ridgebacks (representing at least eight original African and eight Thai female lineages) were analysed and compared to 688 dogs from other breeds. The Rhodesian Ridgebacks were seven dogs from the USA representing seven female lines tracked back to African imports, three dogs from Sweden, and one from Germany. Fifty-two samples were sequenced for this study (Supplementary Table 2), one African Basenji (AY656737) was from GenBank, while 654 samples were from Savolainen et al. 2. Geographic origin of samples: Europe (n=207), Africa (n=69), SW Asia (n=90), East Asia (n=262), India (n=30), Siberia (n=24), and Arctic America (n=25); geographic origin alluding to the historical origin of a breed, even if sampled in another region. Real-time PCR. Dermis samples (dorsal neck) extracted from six Rhodesian Ridgebacks were included in the TaqMan analysis. Two individuals from each genotype were represented; homozygous ridged (R/R), heterozygous ridged (R/r), and homozygous ridgeless (r/r). In an attempt to identify FGF mrna expression, DS- and testis tissue from a four week 3
old Rhodesian Ridgeback were also analyzed. The two R/R and the two R/r individuals were adult dogs and the two r/r individuals were 2 weeks respectively 6 weeks old. cdna was synthesized with the iscript Select cdna Synthesis Kit (BIO RAD) using random primers (supplied) and purified with BD Chroma Spin-10 columns (BD Biosciences). Primers and probes (Supplementary Table 1) were designed with the Primer Express software v 1.5 (Applied Biosystems) and probes were labelled with FAM fluorophore as reporter dye. TaqMan 1000 Rxn Gold/Buffer A was used for master mix and reactions were run on a 7700 Sequence Detector (Applied Biosystems) according to supplier s instructions. All results were analyzed using the Sequence Detection System (SDS) software v 1.9.1. The FGF19 primers were used to amplify genomic DNA to confirm their proper design. β-actin was used as control. Accession numbers. Sequence data reported in this paper have been submitted to DDBJ/EMBL/GenBank with the following accession numbers AM778723-AM778732. References 1. Isaksson, M. et al. MLGA - a rapid and cost efficient assay for gene copy-number analysis. Nucleic Acids Res., in press 2. Savolainen, P. et al. Genetic evidence for an East Asian origin of domestic dogs. Science 298, 1610-1613 (2002). 4
Supplementary Note Phylogenetic analysis. The genetic relationship between Rhodesian and Thai Ridgeback dogs was investigated by sequencing 582 bp of the mitochondrial DNA control region (Supplementary Fig. 2). The analysis did not reveal a close relationship between the two breeds. Except for haplotype A11, which occurs universally and is found in 15% of all dogs in the global population, there was no overlap between the two breeds. The Rhodesian Ridgebacks had haplotypes typical for European and African dogs whereas the Thai Ridgebacks had haplotypes typical for East Asia, five out of seven found exclusively in South and East Asia. Furthermore, resequencing of 2.4 kb just flanking the duplicated region on chromosome 18 revealed multiple sequence differences between Ridge haplotypes from Rhodesian and Thai Ridgebacks (Supplementary Table 3). This result is consistent with our finding that SNP_51,399,353, located at the proximal end of the duplicated region, was monomorphic among Thai Ridgebacks whereas 43 of 45 ridged Rhodesian Ridgebacks were heterozygous for the SNP. Nevertheless, the Ridge allele in the two breeds is most certainly of common origin since they share an identical duplication breakpoint, which does not contain any repetitive sequences expected to promote recombination events. Taken together these results demonstrate that the sharing of the same Ridge mutation between Rhodesian and Thai Ridgebacks is not due to a recent introgression from one breed to the other. Real-time PCR. Real-time PCR and cdna representing dermis (dorsal neck) samples from ridged and ridgeless Rhodesian Ridgebacks were used to study whether the duplication leads to altered mrna expression in post-natal tissue. We were unable to detect mrna expression of FGF3, 4 or 19 in postnatal tissue consistent with their embryonic expression. However, expression of ORAOV1 mrna was about two-fold higher in homozygous ridged dogs compared with ridgeless dogs, consistent with a chromosome duplication (Supplementary 5
Fig. 3). Weak expression of CCND1 was also detected, but no significant difference between genotypes was indicated. 6
Supplementary Table 1. (A) Selector probes and primer sequences used for MLGA analysis. (B) Primer pairs used for PCR analysis and sequencing of the duplication breakpoint. (C) Primer pairs used for PCR analysis and sequencing the upstream flanking region. (D) Primers and probes used for real time-pcr analysis of genes located in the duplicated region in Ridgeback dogs and the β-actin control amplicon. Name Primer/Probe Sequences Pos.Start Pos.Stop Size Chr A. MLGA analysis P51390884* CGCGGGCCAGAGCATCATACGATAACGGTAGAAAGCTTTGCTAACGGTCGAGGTGATGTAGGAGCAGAATTG 51,390,884 51,391,076 193 18 P51393290* CACCTACTTTCCTACTGCATACGATAACGGTAGAAAGCTTTGCTAACGGTCGAGGGTGTGTGTAGCAGGCACC 51,393,290 51,393,503 214 18 P51396781* TCTTGCACTGCGACACCATACGATAACGGTAGAAAGCTTTGCTAACGGTCGAGGGAGCCCCTAAAACAGAGCT 51,396,781 51,396,958 178 18 P51399619* CCACAGCCCCAGGGGCATACGATAACGGTAGAAAGCTTTGCTAACGGTCGAGGTACACCCAAGCTGGGGG 51,399,619 51,399,737 119 18 P51405038* TAACCCAAATAGAGCAGCATACGATAACGGTAGAAAGCTTTGCTAACGGTCGAGGGAGGCCAGGGGTCTACT 51,405,038 51,405,282 245 18 P51453325* ACCCGCAGAGCAGCGCATACGATAACGGTAGAAAGCTTTGCTAACGGTCGAGGTATTCATTGTTGCTCAAGC 51,453,325 51,453,430 106 18 P51483354* TTGATTTTGGCTCAGATCATACGATAACGGTAGAAAGCTTTGCTAACGGTCGAGGACTGGTTCAATGCGTGGAG 51,483,354 51,483,491 138 18 P51523631* TTACCTGGCAAGCCACCATACGATAACGGTAGAAAGCTTTGCTAACGGTCGAGGGACAGCAGGTGCAGGGG 51,523,631 51,523,737 107 18 P51531033* ACTGCCAGCTGCCACCATACGATAACGGTAGAAAGCTTTGCTAACGGTCGAGGTACTCGATCACTTACCGCA 51,531,033 51,531,168 136 18 P51532437* CACGTGCACACACTCTTCATACGATAACGGTAGAAAGCTTTGCTAACGGTCGAGGTGCACACCCACACTCCTT 51,532,437 51,532,500 64 18 P51546495* CTCTAAAGGGTAAACATCATACGATAACGGTAGAAAGCTTTGCTAACGGTCGAGGACTCTGACAGAGGAAGGCA 51,546,495 51,546,646 152 18 P51549178* AGTGGTTGAAAGAATCCCATACGATAACGGTAGAAAGCTTTGCTAACGGTCGAGGACCTGCATCTTAGGAGGCA 51,549,178 51,549,402 225 18 P50121173* AGACCACGCCGACCCCATACGATAACGGTAGAAAGCTTTGCTAACGGTCGAGGACAGCTAGCTGCCATTTCT 50,121,173 50,121,334 162 13 P24857191* CTGTGGCATTTCTAATCCATACGATAACGGTAGAAAGCTTTGCTAACGGTCGAGGCTTTGCGAAAGTTCTACAG 24,857,191 24,857,272 82 20 Vector CTCGACCGTTAGCAAAGCTTTCTACCGTTATCGT Primer Fwd. AGCTTTGCTAACGGTCGAG Primer Rev. AGCTTTCTACCGTTATCGT B. Resequencing of the duplication breakpoint P1 Fwd. CTCGTGGGAGGGCAGCAGTGGGTCCATC 51,398,920 Rev. GCTGAGCCCAGCACCTCCCACACTTGTC 51,531,824 P2 Fwd. AGGACAGGAAGCTGTTGGAA 51,531,096 Rev. CCAAGCTGCCCTTAATCTTG 51,399,491 P3 Fwd. TACTCGAGCCTTGGGAACAT 51,531,515 Rev. CCAAGCTGCCCTTAATCTTG 51,399,491 P4 Fwd. AGGACAGGAAGCTGTTGGAA 51,531,096 Rev. GCTCCCATGTTGACTGGTT 51,399,067 P5 Fwd. TACTCGAGCCTTGGGAACAT 51,531,515 Rev. GCTCCCATGTTGACTGGTT 51,399,067 C. Resequencing of the up-stream flanking region P6 Fwd. CTCTGATGCTGAGGGGAGAG 51,397,907 Rev. TTGATCTGCCAAAACTCGTG 51,398,603 P7 Fwd. CAGCTCTTTCCCAGTTCCTG 51,397,193 Rev. TTGCAGATTGCTTAGGTGGA 51,397,954 P8 Fwd. GCACAACCCTGAATTTGCTA 51,396,512 Rev. TCAGGGCAGAGACTCCTTTG 51,397,270 P9 Fwd. GGTTACATTGCCACCGAACT 51,395,829 Rev. CGCCAACCAGCTTGTTTTTA 51,396,602 7
D. Real-time PCR FGF3 Fwd. CCACGAACTCACACTCGGC 51,414,043 18 Probe TTGTAGCTCTCCGAAGCGTAGAGCCG 51,409,304 18 Rev. GTACCTGGCCATGAACAAGAGG 51,409,279 18 FGF4 Fwd. GCAGGATCTCTTTGAACTTGCA 51,440,988 18 Probe CCTCGGTGAAGAAGGGCGAGCC 51,440,430 18 Rev. TGAGCAGCAAGGGCAAGC 51,440,407 18 FGF19 Fwd. ACGGATCTCCTCCTCGAAAGTAC 51,494,430 18 Probe TCCGGCGGAGTACTGAGGCAGC 51,491,499 18 Rev. CCGACGGCAGGATGCA 51,491,480 18 ORAOV1 Fwd. AGGAAGACAGTATGGCACGTTACA 51,509,491 18 Probe CAAAATTGGATCGGAGATTGGGTGCTATC 51,509,521 18 Rev. CAAGCGAAAGCAAAGCCC 51,511,570 18 CCND1 Fwd. CCGCCTCACTCGGTTCC 51,531,075 18 Probe TCCAAAGTGATTAAGTGTGATGCGGACTGTC 51,531,072 18 Rev. CTCCTGGCACGCCCG 51,528,289 18 β-actin Fwd. CATGGATGCCGCAGGATT 15,434,075 9 Probe TGCCCAGGAAGGAAGGCTGGAAGAG 15,434,054 9 Rev. GTTCCGCTGCCCAGAGG 15,433,921 9 *Selector probes 8
Supplementary Table 2. Haplotype, breed, and geographic information for Rhodesian Ridgebacks, Thai Ridgebacks, and for African and Indian samples analyzed for this study, information for the 654 other dogs are given in Savolainen et al. 2 Haplotype Individual Breed Sub subregion Subregion Region A2 z392 Rhodesian Ridgeback S. Africa Africa A11 L43 Rhodesian ridgeback S. Africa Africa A11 m424 Rhodesian Ridgeback S. Africa Africa A11 m565 Rhodesian Ridgeback S. Africa Africa A11 z393 Rhodesian Ridgeback S. Africa Africa A11 z394 Rhodesian Ridgeback S. Africa Africa A11 z695 Rhodesian Ridgeback S. Africa Africa A24 z390 Rhodesian Ridgeback S. Africa Africa A24 z391 Rhodesian Ridgeback S. Africa Africa B1 z652 Rhodesian Ridgeback S. Africa Africa B1 z656 Rhodesian Ridgeback S. Africa Africa A6 R30 Thai ridgeback Thailand Southeast Asia East Asia A7 P67 Thai ridgeback Thailand Southeast Asia East Asia A8 P63 Thai ridgeback Thailand Southeast Asia East Asia A11 P64 Thai ridgeback Thailand Southeast Asia East Asia A18 P65 Thai ridgeback Thailand Southeast Asia East Asia A35 R29 Thai ridgeback Thailand Southeast Asia East Asia A35 z639 Thai Ridgeback Thailand Southeast Asia East Asia A44 z701 Thai Ridgeback Thailand Southeast Asia East Asia A3 z48 Basenji Benin S. Africa Africa A5 z28 Basenji D.R.Congo S. Africa Africa A5 z32 Basenji D.R.Congo S. Africa Africa A11 m428 Azawakh Mali N. Africa Africa A11 m354 Azawakh N. Africa Africa A11 m358 Azawakh N. Africa Africa A11 m347 S. Africa Africa A16 z46 Basenji Benin S. Africa Africa A16 z74 Lesotho S. Africa Africa A20 z76 Lesotho S. Africa Africa A22 z71 Lesotho S. Africa Africa A22 z72 Lesotho S. Africa Africa A22 z25 South Africa S. Africa Africa A32 z73 Lesotho S. Africa Africa A32 z75 Lesotho S. Africa Africa A127 m351 Azawakh N. Africa Africa A127 m357 Azawakh N. Africa Africa A153 z24 South Africa S. Africa Africa A157 z47 Basenji Benin S. Africa Africa A164 AY656737 Basenji S. Africa Africa B2 m346 Tanzania S. Africa Africa B10 z23 Africanis South Africa S. Africa Africa C3 z27 Africanis South Afr/Mozamb S. Africa Africa D7 m353 Azawakh N. Africa Africa A11 m4 India India A11 m6 India India A11 m7 India India A11 m10 India India A11 m208 Tamil Nadu India India A16 m206 Tamil Nadu India India A16 m207 Tamil Nadu India India A20 m204 Tamil Nadu India India A76 m200 Tamil Nadu India India A77 m211 Tamil Nadu India India A78 m212 Tamil Nadu India India B1 m202 Tamil Nadu India India B1 m205 Tamil Nadu India India C3 m8 India India C3 m201 Tamil Nadu India India 9
C3 m203 Tamil Nadu India India C5 m209 Tamil Nadu India India 10
Supplementary Table 3. Results of resequencing the region from 51,396,118 bp to 51,398,557 bp on dog chromosome 18 from homozygous ridge (R/R) and ridgeless (r/r) dogs. The reference sequence (Boxer) is from the genome assembly. Dash (-) indicates sequence identity to the reference; blank indicates missing data. The primers used for resequencing are given in Supplementary Table 1. Nucleotide position 51396325 Breed Genotype Boxer r/r C C 0 C A T A G Rhodesian Ridgeback R/R - -/T - - - - - - Rhodesian Ridgeback R/R - - - - - Thai Ridgeback R/R T T C G G C G - Thai Ridgeback R/R T T C G G C G A/- Rhodesian Ridgeback r/r -/T -/G -/G C/- -/G A/- Rhodesian Ridgeback r/r -/T T -/G -/G C/- -/G A/- Basenji r/r -/T T 0/C -/G -/G C G A/- Basenji r/r -/T T 0/C -/G -/G C G A/- * Insertion/deletion polymorphism, 0=no insertion 51396717 51397075* 51397693 51397696 51397705 51397780 51397822 11
Hillbertz et al. Supplementary Figure 1 51,398,486 51,398,518 51,398,548 5' BP BP 3' BP 51,531,911 51,531,941 51,531,973 Supplementary Figure 1. Alignment of the sequence at the breakpoint (BP) between the first and second copy of the duplication (middle) and the corresponding sequences at the 5 (top) and 3' (bottom) ends of the duplicated region.
Hillbertz et al. Supplementary Figure 2 30 70 58 62 30 70 58 62 42 43 1 71 17 10 33 5 16 2 74 9 7 8 76 6 157 56 4 3 22 46 23 42 43 1 10 17 5 16 2 74 9 71 33 7 8 3 76 6 157 56 4 22 46 23 53 39 45 72 38 55 75 60 73 59 35 52 24 49 31 29 77 78 34 25 127 44 15 11 51 153 32 14 41 12 26 40 18 54 47 28 20 27 36 65 19 66 63 68 67 21 64 69 164 53 39 45 72 38 55 75 60 73 59 35 52 44 29 15 78 153 14 41 18 47 28 27 36 20 66 19 63 68 69 49 11 67 21 34 24 51 12 40 54 26 65 25 127 32 31 77 64 164 Supplementary Figure 2. Striking differences in mtdna haplotype composition between Rhodesian Ridgebacks (RR) and Thai Ridgebacks (TR). The minimum spanning network was constructed using Arlequin (version 2.000) and shows haplotypes of the major clade A, in which all TR and all but two RR haplotypes fall. Haplotypes (open circles) and empty nodes (solid dots) are separated by one substitutional step. Haplotype names, without the prefix A, are given by the numbers in the circles. Left network: colored circles indicate haplotypes found in RR and/or in other dogs from the western part of the world (Europe, Africa and SW Asia; 362 dogs); green for haplotypes found in RR (Type A2 found in one RR, type A11 in six RR, type A24 in two RR) as well as other dogs. Right network: colored circles indicate haplotypes found in TR and/or in dogs from East Asia, India, Siberia, and Arctic America; 341 dogs); orange for haplotypes found in TR (Type A35 in two TR, one TR for each of the other haplotypes; haplotypes A6, A7 and A8 are unique to TR). Violet lines highlight East Asian-specific parts of the network. Two alternative links (two steps between haplotype 35 and 153, and three steps between haplotype 41 and 42) were not drawn in order to simplify the figure.
Hillbertz et al. Supplementary Figure 3 4 β-actin vs. ORAOV1 5 Delta CT values 6 7 8 9 10 r/r R/r R/R Genotypes Supplementary Figure 3. Real-time PCR analysis of ORAOV1 using cdna from dermis representing two ridgeless (r/r) and four ridged (two R/r and two R/R) Rhodesian Ridgebacks. Delta CT values (mean ± SE based on triplicates) obtained for β-actin ORAOV1 are presented. There is a statistically significant positive correlation between ORAOV1 copy number and its relative expression (P = 0.05).