A functional variant of the dopamine D3 receptor is associated with risk and age-at-onset of essential tremor -- Jeanneteau et al. 103 (28): 10753 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Jeanneteau et al. 10.1073/pnas.0508189103.

Supporting Information

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Supporting Table 2
Supporting Table 3
Supporting Table 4
Supporting Figure 5
Supporting Figure 6
Supporting Figure 7

Fig. 5.
Neuronal localization of dopamine D₃ receptor (DRD3) variants. (A) DRD3 variants were transfected with Lipofectamine 2000 (Invitrogen) in embryonic day (E) 18 cortical primary cultures from rat brain at 10 days in vitro (DIV), and expression was allowed for 48 h in neurobasal (Invitrogen) medium supplemented with 2% B27 (Invitrogen). Images are composite of several confocal stack fluorescence images captured by a laser scanning confocal image system (Leica, Deerfield, IL). Both variants yellow fluorescent protein (YFP)-tagged at the C terminus displayed the same localization in the soma and projections without apparent discrepancies. The Ser-9 variant fused to GFP at the N terminus, used as a control, showed the same distribution. Shown is epifluorescent detection of YFP-tagged variants and DARPP32 (1/200; Santa Cruz Biotechnology ) (B) or synaptophysin (1/500; Sigma) (C) in E18 striatal primary cultures from rat brain. Neuronal cultures were transfected with Lipofectamine 2000 at 6 DIV, and expression was allowed for 48 h in neurobasal supplemented with 2% B27. Both variants are mostly localized in the soma and to a lesser extent in projections where both variants partially colocalized with synaptophysin.

Supporting Figure 6

Fig. 6.
DRD3 variants anterograde trafficking. human embryonic kidney (HEK) 293 cells were transiently transfected with Lipofectamine 2000 with each YFP-tagged variant, and expression was allowed overnight. As outlined in A, surface proteins were first blocked, and newly inserted proteins were labeled with biotin. Briefly, cells were rinsed three times with PBS containing 1 mM CaCl₂ and 0.5 mM MgCl₂ (PBS-Ca-Mg) and incubated with 1.5 mg/ml sulfo-NHS-acetate (Pierce) in PBS for 30 min at 4°C. Reaction was quenched for 20 min at 4°C by using 100 mM glycine dissolved in TBS. Cells were subsequently incubated at 37°C for the indicated time. Surface proteins were then biotinylated with 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) dissolved in PBS for 30 min at 4°C. Unreacted biotin was quenched with PBS-Ca-Mg containing 100 mM glycine for 20 min at 4°C. Cells were then lysed in RIPA buffer (1% Nonidet P-40/10% glycerol/150 mM NaCl/1 mM EDTA/10 mM Tris, pH 8.0/10 mM NaF/1 mM sodium orthovanadate/2 mg/ml aprotinin/1 mg/ml leupeptin/25 mg/ml phenylmethylsulfonyl fluoride) for 30 min on ice. Lysates were clarified, and equal biotinylated proteins were pulled-down with streptavidin-agarose (Pierce). Immobilized proteins were rinsed five times with RIPA buffer and resolved in 10% SDS/PAGE. (B) DRD3 variants were detected in the pull-down and input samples by an anti-GFP antibody (Sigma). Densitometric analysis was performed using the NIH IMAGE 1.63 software (http://rsb.info.nih.gov/nih-image/). GFP intensities in the purified biotinylated proteins were divided by total GFP intensities from inputs to determine the ratio (in bold) of newly inserted DRD3 variants. Experiments and blots are representative of two independent experiments.

Supporting Figure 7

Fig. 7.
Retrograde trafficking of DRD3 variants. HEK293 cells were transiently transfected with Lipofectamine 2000 by GFP-tagged DRD2 or each YFP-tagged DRD3 variant, and expression was allowed for 48 h. As outlined in A, surface proteins were first labeled with cleavable biotin; then, constitutive and dopamine-induced internalization was allowed for 90 min at 37°C, followed by shedding of biotin from residual surface proteins. Briefly, cells were rinsed three times with PBS and incubated with 0.3 mg/ml sulfo-NHS-SS-biotin (Pierce) in PBS for 30 min at 4°C. Reaction was quenched for 20 min at 4°C by using 100 mM glycine dissolved in PBS. Cells were subsequently incubated at 37°C in the presence or absence of 10 mM dopamine for the indicated times. Assays were stopped by chilling cells and washing three times with ice-cold PBS. Cleavage of surface-localized biotinylated proteins was achieved with two changes of glutathione strip buffer (50 mM glutathione/75 mM NaCl/75 mM NaOH/10% FBS in H₂O) for 20 min each at 4°C (1) . Excess glutathione was then quenched for 30 min at 4°C in iodoacetamide buffer (50 mM iodoacetamide/1% BSA in PBS, pH 7.4). Cells were then lysed in RIPA buffer and clarified, and equal biotinylated proteins were pulled-down with streptavidin-agarose. Immobilized proteins were rinsed five times with RIPA buffer and resolved in 10% SDS/PAGE. (B) DRD2 and DRD3 subtypes were detected in the pull-down and input samples by an anti-GFP antibody. Endogenous transferrin receptor (Tfr) was detected with a Tfr antibody (Zymed) as an internal control. Experiments and blots are representative of three independent experiments. (C) The same procedure was used to compare retrograde trafficking between Ser-9 and Gly-9 variants. Experiments and blots are representative of four independent experiments. (D) Densitometric analysis was performed using the NIH IMAGE 1.63 software. GFP intensities were normalized to inputs and compared with total biotinylated proteins that were not subjected to the stripping procedure to finally determine percentages of DRD3 variants internalized. n.d., not determined.

1. Vickery, R. G. & Von Zastrow, M. (1999) J. Cell Biol. 144, 31-43.

Table 2. Results of the parametric linkage analysis

Model used	Mutation rate	Phenocopy rate	LOD score at θ = 0	LOD score at θ = 5 cM
Model 1	0.01	0.01	7.99	7.00
Model 2	0.02	0.05	7.13	6.23
Model 3	0.05	0.10	6.25	5.44
Model 4	0.35	0.05	6.38	5.54
Model 5	0.50	0.10	5.38	4.65

All calculations were performed with the GENEHUNTER program (1), assuming an autosomal dominant mode of inheritance with a penetrance of 0.90. Various values of mutation and phenocopy rates were tested in the analysis. LOD, logarithm of odds.

1. Kruglyak, L., Daly, M. J., Reeve-Daly, M. P. & Lander, E. S. (1996) Am. J. Hum. Genet. 58, 1347-1363.

Table 3. Haplotype block containing the DRD 3 Ser9Gly variant

dbSNP ID	MAF	Chromosomal position	D ' value with marker:
dbSNP ID	MAF	Chromosomal position	rs167770	rs6798102
rs167771	0.17	115,197,176	0.52	0.42

rs167770	0.29	115,200,463	NA	1.00
rs324029	0.29	115,202,524	1.00	1.00
rs10934256	0.21	115,206,553	1.00	0.94
rs1486009	0.06	115,209,433	1.00	0.80
rs6798102	0.27	115,251,348	1.00	NA

rs1503673	0.15	115,268,764	0.76	0.52

MAF: minor allele frequency; NA, not applicable; HB, haplotype block; LD, linkage disequilibrium. LD around the DRD3 Ser9Gly variant was studied by using HapMap haplotype data (www.hapmap.org). We defined a 1-Mb interval of interest, centered on the DRD3 gene, and retrieved genotypic data from 125 single nucleotide polymorphisms (from rs12630791, chromosomal position: 114,708,618, to rs9820450, chromosomal position: 115,697,886). Long distance LD and haplotype analyses were performed by using HAPLOVIEW 2.05 software (www.broad.mit.edu/personal/jcbarret/haplo/index.php).

The DRD3 Ser9Gly variant (named rs6280 in dbSNP, chromosomal position 115,211,716) is located in an HB of ≈50 Kb, as defined in ref. 1, bordered by markers rs167770 and rs6798102 on each side. Shown are LD values (D') between HB boundaries (rs167770 and rs6798102) and each HapMap marker in the HB (bold type). LD values with the closest markers outside the HB (rs 167771 and rs1503673) are provided for comparison. Use of other definitions of HB ("four gametes rule" as described in ref. 2, or "solid spine of LD", as defined in Haploview documentation) did not change the HB boundaries. Because the DRD3 Ser9Gly polymorphism was not included in HapMap genotyped markers, we "anchored" this variant to the haplotype map by genotyping rs6280 and rs167770 in 172 unrelated controls from the general population. Both markers were genotyped by the TaqMan method, on an ABI Prism 7000 Sequence Detection System (Applied Biosystems; TaqMan SNP genotyping assays ID: C_949770_10 for rs6280 and C_3134171_10 for rs167770, both available from Applied Biosystems). Both markers were in complete linkage disequilibrium (D' = 1.00; rs6280 MAF: 0.31, rs167770 MAF: 0.24), confirming that rs6280 belongs to this HB.

1. Gabriel, S. B., Schaffner, S. F., Nguyen, H., Moore, J. M., Roy, J., Blumenstiel, B., Higgins, J., DeFelice, M., Lochner, A., Faggart, M., et al. (2002) Science 296, 2225-2229.

2. Wang, N., Akey, J. M., Zhang, K., Chakraborty, R. & Jin, L (2002) Am. J. Hum. Genet. 71, 1227-1234.

Table 4. Variations identified in DRD3 genomic sequence

Target sequence	Target boundaries	Amplification boundaries	Variations identified
Promoter	- 7759 to -7146*	- 8017 to -7318, -7585 to -6926	None
Exon 1	- 7145 to -6665*	- 7215 to -6515	G-6734A (5' untranslated) : P.24 AG, P.26 AA.
Exon 2	- 35 to +270 (Start codon: +1)	- 229 to +465	A25G (Ser9Gly): P.21 GG, P.25 GG, P.26 GG, P.27 AG, P.29 AG, P.30 AG; A51G (Ala17Ala): P.25 GG.
Exon 3	+12126 to +12238	+11945 to +12456	T12373C (intronic) : P.29 TC.
Exon 4	+24436 to +24578	+24203 to +24791	T24289C (intronic) : P.26 CC.
Exon 5	+32297 to +32493	+32055 to +32639	A32490G (Gln240Gln) : P.25 GG; C32577T (intronic): P.26 TT.
Exon 6	+40593 to +40875	40446+ to +41092	A40856G (Gln329Gln) : P.25 GG.
Exon 7	+43081 to +43340^† (Stop codon: +43275)	+42856 to +43503^†	None

*As defined in ref. 1.

^†As defined in ref. 2.

We sequenced PCR fragments encompassing the genomic sequence of DRD3 exons and promoter (reference sequence: Homo sapiens chromosome 3 genomic contig, Genbank accession NT_005612) in nine probands from families 21, 22, 23, 24, 25, 26, 27, 29, and 30. All nucleotide positions are given with respect to the DRD3 translation start site (in exon 2). P.21, P.22, etc. designate probands of families 21, 22, etc. and are followed by the nucleotides sequenced on their two chromosomes; only homozygous or heterozygous carriers of the low-frequency allele are mentioned for each nucleotide variation. Primers used for amplification and sequencing were the following:

-promoter region: gDRD3prom2F: 5'-CCTCCATAAGCTTTAATTTTCCT-3', gDRD3prom2R: 5'-GGAGGTGTAAGCCTGTCTGG-3'

gDRD3prom1F: 5'-TGTCCTTGGTTGCATCTCAC-3', gDRD3prom1R: 5'-GGGAGGGACCCTAAACTGAG-3'

-exon 1: gDRD3ex1F: 5'-ACACCCACCAAAATCTGGAA-3', gDRD3ex1R: 5'-AACCATTTCCTCAGGGCTTT-3'

-exon 2: gDRD3ex2F: 5'-ATGGGAGCTTCAAAGGGAAG-3', gDRD3ex2R: 5'-TCTGATGTGGATGAGGGACA-3'

-exon 3: gDRD3ex3F: 5'-GGCAACCAGTTTTGGATAGC-3', gDRD3ex3R: 5'-AGAAGAAAGGCCAGGAAAGC-3'

-exon 4: gDRD3ex4F: 5'-ATAATTAGGCAGGCGTGGTG-3', gDRD3ex4R: 5'-CTTTACCCCCTGTGCCAATA-3'

-exon 5: gDRD3ex5F: 5'-CCCAGTGTCTGGCGTATGAT-3', gDRD3ex5R: 5'-CGCAGCCAAAATTAGCAGAT-3'

-exon 6: gDRD3ex6F: 5'-CCACCACTCCCTCAGATGTT-3', gDRD3ex6R: 5'-AGCGCAGTTAAGTGGCTTGT-3'

-exon 7: gDRD3ex7F: 5'-CTCCTGACCTCAGGTGATCC-3', gDRD3ex7R: 5'-GCTCCCCAGTCATTTGAAGA-3'

We found no other nonsynonymous mutations than the Ser9Gly variant in the coding sequence. All other nucleotide variations were rare and synonymous, noncoding, or intronic and were not present in the splicing junctions. If one assumes that the disease-causing mutation has the frequency higher than 70 % in the families, the probability to detect it in nine patients is higher than 99.99998%.

1. Anney, R. J., Rees, M. I., Bryan, E., Spurlock, G., Williams, N., Norton, N., Williams, H., Cardno, A., Zammit, S., Jones, S., et al. (2002) Mol. Psychiatry 7, 493-502.

2. Giros, B. Martres, M. P., Sokoloff, P. & Schwartz, J.C. (1990) C. R. Acad. Sci. III 311, 501-508.