humans. Comparison of the genomic organization of the overlapping

(1)

Figure 1. Genomic organization of ATXN8OS, ATXN8, and KLHL1 in

humans. Comparison of the genomic organization of the overlapping

ATXN8 and ATXN8OS genes and 5'-region of the KLHL1 gene. Exons are

shown as boxes and various alternative splice forms are indicated by

dashed lines. Also indicated are amplified regions for (CTG)

_n

genotyping,

intron C1 ~ intron B and intron B ~ exon A downstream sequencing

(black bars), as well as the locations of SNP1 T/C and SNP2 C/T

(asterisks).

(2)

Figure 2. The schematic diagram of luciferase reporter constructs. The

ATXN8/ATXN8OS promoter fragments were inserted upstream of the

firefly luciferase reporter gene at the EcoRI site added between the

HindIII/XhoI sites of the pGL3-basic vector (Hou, 2005).

(3)

Figure 3. Frequency distributions of the SCA8 repeat lengths in controls and patients with PD, dementia, tremor, or other neurological disorders.

The percentages of the large alleles (85 ~ 120 repeats) (A) or genotypes

(B) in each group are shown in parentheses.

(4)

Figure 4. SNPs identification. (A) Location of the two novel SNPs.

SNP1 is 157 nucleotides downstream from the 5' donor splice site of

exon B, whereas SNP2 is 64 nucleotides downstream from the CTG

repeats in exon A. (B) DNA sequence analysis of the SNP1 T/C and

SNP2 C/T.

(5)

Figure 5. Restriction enzyme and haplotype analysis of the SNP1 and SNP2. (A) The PCR products containing SNP1 (left) or SNP2 (right) were digested with Cfr13I or Mph1103I and resolved on a 2% agarose gel. Lane M (HinfI digest of pGEM4 DNA) is the size marker. (B) Pedigree and haplotype analysis of H600 and H327 families. Haplotypes for SNP1, CTG repeat number and SNP2 are shown under each person.

Haplotypes carrying the expanded CTG repeats are boxed.

(6)

Figure 6. Transient transfection of HEK-293, SK-N-SH and IMR-32

cells with ATXN8/ATXN8OS promoter constructs monitored by

fluorescence microscope (A) and (B) flow cytometry.

(7)

Figure 7. Deletion analysis of the ATXN8 promoter. (A) Schematic

representation of DNA constructs containing ATXN8 promoter region

(-1035~-6, -518~-6 and -99~-6) linked to the firefly luciferase reporter

gene. The BsaBI and DraI restriction sites were used to generate different

lengths of promoter fragments. The translation initiation site (AF252279,

nucleotide 23763) is used as temporary transcription start site +1. (B)

Transient expression of luciferase activity driven in HEK-293, SK-N-SH

and IMR-32 cells. The Renilla luciferase plasmid (phRL-TK, as an

internal control) was co-transfected with each construct. The relative

luciferase activity represents the ratio of the firefly luciferase level to the

Renilla luciferase level. Data are represented as the mean ± SD of three

independent experiments, each performed in triplicate. The * indicates

the significant difference between comparisons.

(8)

Figure 8. Nucleotide sequences (-99~+61) of ATXN8 proximal promoter region and polyglutamine coding open reading frame. Three TATA boxes predicted (-100~-95, -53~-48 and -42~-37) (http://gpminer.mbc.nctu.

edu.tw/index.php) are indicated. Putative transcription factors binding

motifs (http://mbs.cbrc.jp/research/db/TFSEARCH.html) with significant

homolog for GATA-1 (score 90.3), C/EBPβ (score 83.6) and S8 (score

85.8) are marked in green, purple and yellow, respectively. The translation

initiation site is noted as temporary transcription start site +1.

(9)

Figure 9. C/EBPβ cDNA co-transfection and luciferase reporter assay.

The Renilla luciferase plasmid (phRL-TK) was co-transfected with the

ATXN8 proximal promoter construct (-99~-6) containing -62G or -62A into

HEK-293, SK-N-SH and IMR-32 cells and the relative firefly/Renilla

luciferase activity (A), ratio (+/- C/EBPβ) (B) and ratio (-62G/-62A) (C)

were measured. Data are represented as the mean ± SD of three

independent experiments, each performed in triplicate. The * indicates

the significant difference between comparisons.

(10)

Figure 10. RT-PCR analysis of transfected ATXN8OS promoter constructs. (A) Schematic diagrams of ATXN8OS promoter (white box), exon D (dotted box) and luciferase reporter (black box). The approximate location of alternative splice donor sites within exon D is indicated.

Transcription start sites of exon D5 (Benzow and Koob, 2002) and exon

D (NR_002717) are represented by +1 and +801, respectively. Also

indicated are primers used for RT-PCR detection of ATXN8OS-luciferase

mRNA. (B) Agarose gel (1.0 ~ 1.4%) electrophoresis of RT-PCR

amplified products of pGL-3 vector (lane V) or ATXN8OS promoter

constructs (-1253~+800 and -114~+800)-transfected HEK-293 (lane H)

and SK-N-SH (lane S) cells using primers F1~F3 and R. Lane M (HinfI

digest of pGEM4 DNA) was used for size markers.

(11)

Figure 11. RT-PCR analysis of ATXN8OS expression in HEK-293,

SK-N-SH and IMR-32 cells. (A) Schematic diagrams of ATXN8OS

promoter and exon D region with alternative splice donor sites and

transcription start sites of exon D5 (+1) and exon D (+801) as described

(Fig. 10). Also indicated are primers used for RT-PCR detection of

ATXN8OS mRNA. (B) Agarose gel electrophoresis of amplification

products using primers F1~F4 and R1. (C) Agarose gel electrophoresis

of amplification products using primers F5/F6 and R2. β-Actin PCR was

included as quality control of cDNA preparation. Lane M (HinfI digest

of pGEM4 DNA) was used for size markers.

(12)

Figure 12. (A) Schematic diagrams of pEF-ATXN8OS (carrying 23, 88 and 157 CRs) constructs. (B) Ribonuclear foci formation with ATXN8OS CUG expansion. HEK-293 cells were transfected with ATXN8OS cDNA constructs carrying 23, 88 or 157 CRs and analyzed 48 hr later by RNA-FISH using a Cy3 labeled (CAG)

10

(green, upper panel) or Cy5 labeled ATXN8OS unique sequence (red, lower panel) oligonucleotide probe. (C) Co-localization of ribonuclear foci with ubiquitin and HSP70.

HEK-293 cells were transfected with ATXN8OS constructs carrying 157

CRs and analyzed 48 hr later by immunofluorescence-FISH using a

(CAG)

₁₀

probe (green) and polyclonal goat anti-ubiquitin or HSP70 and

Cy5 labeled secondary antibodies (red). In both (B) and (C), nuclei were

counterstained with DAPI (blue). (The scale bar = 20 μm)

(13)

Figure 13. Expression of ATXN8OS cDNA constructs in doxycycline

induced (+Dox, 1 µg/ml for 48 h) or non-induced (-Dox) isogenic 293

cells. (A) Annexin V binding in isogenic cells expressing 0, 23, 88, and

157 CTA/CTG combined repeats. The * indicates the difference between

the indicated samples are statistically significant. (B) Flow cytometric

analysis of cell cycle distribution (percent cells) after PI (propidium

iodide)-staining. Data represent the mean and SD (error bar) of three

independent experiments. (C) FACS images of isogenic vector, 23 and

157 CR cells.

(14)

Table 1. Primers for SCA8 genotyping, sequencing and SNP RFLP analysis Test (amplified

region) Primer Anneal (℃) /

MgCl

2

(mM)

Product (bp) or

RFLP enzyme (fragment, bp) Genotyping

(exon A)

3F: fam-GCTTGTGAGGACTGAGAATG 4R: CCCTGGGTCCTTCATGTTAG

49 / 1.5 277~349 (16~40 repeats) Sequencing (intron C1

~ intron B)

F: AGTTGTGGGAGAGATGGGT R: GGAAGAGCAGGGAAGAATG

53 / 1.0 510

Sequencing (intron B

~ exon A downstream) F: CCTAGTTCTTGGCTCCAGA R: GCTTGTGAGGACTGAGAATG

53 / 1.5 484

SNP1 T/C RFLP

analysis F: TATTGGGAGTAAGAACACTGAATGGAC R: AATGTGGATAAGAGATAAGACAAAATTGG

53 / 1.0 Cfr13I: GGNCC* (112 / 88, 24) SNP2 C/T RFLP

analysis F: TGATGTTATAATTGTTATATATTTATGCA R: AACTAACTCAACATCCAGATAATTT

47 / 1.0 Mph1103I: ATGCAT* (128 / 99, 29)

*The restriction site created by PCR using a mismatch primer. The underlines in the primer sequence and enzyme

recognition site indicate the mismatch nucleotide and polymorphic site, respectively.

(15)

Table 2. Primers for SCA8 promoter and CEBPβ cDNA cloning Gene (amplified

region)

Primer Anneal (℃) /

MgCl

₂

(mM)

Product (bp)

ATXN8 (-1035~+10)

F: AACACATGCCCTAAATTTTGG R: GCTGCTGCATTTTTTAAAAATATAT

53 / 2.0 1045

ATXN8 (-99~+10)

F: acctgcctcgagTAAATTCTTAAGTAAGAGATAAGC R: GCTGCTGCATTTTTTAAAAATATAT

54 / 1.0 109

ATXN8OS (-1253~+800)

F: GATTTTGGTTGGTAGATGGGTTAATT R: GTGGACACAGGTACAGTAAGTCCCC

57 / 1.5 2053

CEBPβ cDNA F: TTCATGCAACGCCTGGTGGCCTGGGACC R: TTAAATAACACCACGGGCGGGAGCCCCATCT

68 / 2.0 1272

For ATXN8, the translation initiation site (AF252279, nucleotide 23763) is used as temporary transcription start site +1,

whereas for ATXN8OS, the first nucleotide of exon D5 (reverse complemented strand of AF252279, nucleotide 109586) is

used as transcription start site +1. The lower case letters in the 5' end of ATXN8 forward primer (-99~+10) contains an XhoI

restriction site (underlined) for promoter cloning.

(16)

Table 3. Primers for RT-PCR analysis of endogenous ATXN8OS and transfected ATXN8OS promoter constructs

Amplified region Primer Anneal (℃) /

MgCl

₂

(mM)

Product (bp)

+675~+800 F1: CTCTTAACCCACATTTTCAA

R1: GTGGACACAGGTACAGTAAGTCCCC

54 / 3.0 126

+473~+800 F2: CGGTAGATGACAGAAGAACAC R1: GTGGACACAGGTACAGTAAGTCCCC

54 / 3.0 328

+234~+800 F3: GAGAAGTTGGGACAGAGAAT

R1: GTGGACACAGGTACAGTAAGTCCCC

54 / 3.0 567

+43~+800 F4: GAACTCTACACAAAGAAGCAT

R1: GTGGACACAGGTACAGTAAGTCCCC

54 / 3.0 758

-21~+140 F5: CAAAATGTAAAACTTGGGAGT R2: GATTTGTTGGCTCAGTATTGA

51 / 3.0 161

-62~+140 F6: CAGTAATGATGTGGTTTAGAATAGT R2: GATTTGTTGGCTCAGTATTGA

51 / 3.0 202

+675~+800 plus 265 bp* F1: CTCTTAACCCACATTTTCAA R: CGAAGTACTCAGCGTAAGTG

52 / 2.0 391

(17)

Table 3. Primers for RT-PCR analysis of endogenous ATXN8OS and transfected ATXN8OS promoter constructs (continued)

+473~+800 plus 265 bp* F2: CGGTAGATGACAGAAGAACAC R: CGAAGTACTCAGCGTAAGTG

52 / 2.0 593

+234~+800 plus 265 bp* F3: GAGAAGTTGGGACAGAGAAT R: CGAAGTACTCAGCGTAAGTG

52 / 2.0 832 β-actin

(Internal control) F: GGTCATCACCATTGGCAATG R: TCCTTCTGCATCCTGTCGG

51 / 1.0 305 (gDNA) 210 (cDNA)

*The plus 265 bp represents 5' end of luciferase RNA from transfected ATXN8OS promoter constructs.

(18)

Table 4. Distributions of the SCA8 repeat lengths in controls and patients with PD, dementia, tremor or other neurological disorders.

CTG Controls PD

Dementia

Tremor Others

no. No. % No. % No. % No. %

18 105 25.9 49 23.8 90 26.9 46 23.7 36 22.5

19 1 0.2 2 1.0 4 1.2 1 0.5 1 0.6

20 0 0.0 0 0.0 1 0.3 0 0.0 0 0.0

21 16 3.9 4 1.9 3 0.9 2 1.0 0 0.0

22 6 1.5 2.0 1.0 2.0 0.6 1.0 0.5 5 3.1 23 25 6.2 16.0 7.8 24.0 7.2 13.0 6.7 9 5.6 24 50 12.3 24 11.7 41 12.3 30 15.5 35 21.9 25 32 7.9 23 11.2 31 9.3 15 7.7 16 10.0 26 19 4.7 16 7.8 24 7.2 17 8.8 11 6.9 27 52 12.8 30 14.6 31 9.3 16 8.2 14 8.8 28 47 11.6 12 5.8 43 12.9 18 9.3 10 6.3 29 21 5.2 19 9.2 28 8.4 6 3.1 5 3.1

30 14 3.4 6 2.9 2 0.6 14 7.2 7 4.4

31 9 2.2 1 0.5 3 0.9 6 3.1 6 3.8

32 2 0.5 0 0.0 3 0.9 2 1.0 1 0.6

33 1 0.2 0 0.0 0 0.0 2 1.0 0 0.0

34 1 0.2 0 0.0 1 0.3 2 1.0 1 0.6

35 1 0.2 1 0.5 1 0.3 1 0.5 0 0.0

36 0 0.0 1 0.5 2 0.6 1 0.5 1 0.6

37 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0

38 0 0.0 0 0.0 0 0.0 1 0.5 0 0.0

39 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0

43 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0

85~120 1 0.2 0 0.0 0 0.0 0 0.0 2 1.3

Total 406 206 334 194 160

Mean 24.2 24.1 24.0 24.6 25.2

Std 5.4 4.1 4.3 4.6 10.3

(19)

Table 5. Expanded trinucleotide repeat sequences and numbers in a normal control and patients with Huntington's disease or multiple system atrophy

*In addition to ATXN8OS CTG expansion, huntingtin expansion was also seen in H1608 (43 CAG repeats).

Subject Age/Sex Repeat no. Repeat sequence Diagnosis H1520 61/M 85 (CTA)

₁₀

(CTG)

₈₀

Normal control H1608* 42/M 96 (CTA)

10

(CTG)

86

Huntington's

disease

H1691 51/F 120 (CTA)

10

(CTG)

110

Multiple system

atrophy

(20)

Table 6. Change of CTG expansion during intergenerational transmission in two Parkinson's disease patients

Subjec t

Age/Se x

Expanded repeat no.

Repeat sequence Diagnosis

H327 family

H327 71/F 82 (CTA)

12

(CTG)

70

Parkinson's

disease

H1559 40/F 87 (CTA)

10

(CTG)

77

Normal

H1583 38/M - Normal

H1646 46/F 86 (CTA)

12

(CTG)

74

Normal

H1666 49/F - Normal

H1667 51/M 87 (CTA)

12

(CTG)

75

Normal

H1668 44/F 90 (CTA)

₁₃

(CTG)

₇₇

Normal

H600 family

H600 60/F 92 (CTA)

₇

(CTG)

₂

CTACTGCTA(CTG)

₈₀

Parkinson's disease

H1565 65/F 92 (CTA)

₆

(CTG)

₈₆

Normal

H1566 62/F 88 (CTA)

7

(CTG)

81

Normal

H1574 52/M - Normal

H1575 57/F - Normal

(21)

Table 7. Hardy-Weinberg equilibrium analysis of SCA8 SNP1 T/C and SNP2 C/T

Experimental Expected P-value

No. (%) No. (%) SNP1 T/C

TT 39 38.2 38.3 37.5 TC 47 46.1 48.4 47.5

CC 16 15.7 15.3 15.0 0.958 SNP2 C/T

CC 46 45.1 48.7 47.8 CT 49 48.0 43.5 42.7

TT 7 6.9 9.7 9.5 0.449

(22)

**Table 8. Pairwise linkage disequilibrium measures* for SCA8 SNPs**

D'

SNP1 T/C SNP2 C/T

SNP1 T/C 1.13 0.24

Δ

²

SNP2 C/T 0.02 0.88

*Lewontin's standardized disequilibrium coefficients (D') are given above

the diagonal and the squared pairwise correlations (Δ

²

) are given below

the diagonal; the eigenvalues (λs) associated with the LD correlation

matrix are given along the diagonal (bold, italic).

(23)

Table 9. SNP1/SNP2 and SCA8/SNP2 haplotype distributions

Subjects with SCA8 expansion (n = 15)

Normal control (n = 87)

No. (%) No. (%)

SNP1/SNP2 haplotype

T-C 12.6 42.0 71.4 41.0

T-T* 1.4 4.7 39.6 22.8

C-C 12.4 41.3 44.6 25.6

C-T 3.6 12.0 18.4 10.6

Expanded allele

Normal allele

SNP2 allele No. (%) No. (%)

C 14 (93.3) 11 (73.3)

T 1 (6.7) 4 (26.7)

*Tended to be different (P = 0.041).