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Supplementary Materials: A Comprehensive Characterization of Mitochondrial. Genome in Papillary Thyroid Cancer. Xingyun Su, Weibin Wang, Guodong Ruan, ...

Int. J. Mol. Sci. 2016, 17, 1594; doi:10.3390/ijms17101594

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Supplementary Materials: A Comprehensive Characterization of Mitochondrial Genome in Papillary Thyroid Cancer Xingyun Su, Weibin Wang, Guodong Ruan, Min Liang, Jing Zheng, Ye Chen, Huiling Wu, Thomas J. Fahey III, Minxin Guan and Lisong Teng Table S1. Heteroplasmic mtDNA variations in entire mitochondrial genome. Position

Gene

Change

Amino-Acid Change or Watson-Crick Base-Pairing

2274 3183 3275–3276 14675 14693

16S rRNA 16S rRNA tRNALeu (UUR) tRNAGlu tRNAGlu

A–G T–C Del CA A–G A–G

– – – – –

3392 5977 6238 6596 7104 7822 8005 8156 9253 10573 11032–11038 11673–11677 12418–12425 12540 12794 12858 12943

ND1 COI COI COI COI COII COII COII COIII ND4L ND4 ND4 ND5 ND5 ND5 ND5 ND5

G–A G–A T–C C–T T–C A–G T–C G–A G–A G–A A7–6 C5–C4 Del A A–G T–A Ins T C–T

No: Gly -> Asp No: Trp -> Ter c No: Leu -> Pro Tyr -> Tyr No: Ser -> Pro Pro -> Pro Asn -> Asn No: Val -> Met No: Trp -> Ter No: Gly -> Glu Trp -> Trp No: Leu -> Ter No: Leu -> Phe

Conservation Reported b Index (%) a RNA region 100% N 80.5% N N 61% Y 97.6% Y Protein-coding region 100% Y 100% N 100% N 100% Y 100% N 100% Y 97.6% Y 75.61% Y 100% N 100% Y Y N Y 100% Y 100% N N 36.6% N

Number of 66 PTC Patients (%)

Number of 376 Healthy Controls (%)

Somatic/Germline

1 (1.52%) 1 (1.52%) 1 (1.52%) 2 (3.03%) 2 (3.03%)

0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 6 (1.6%)

Somatic Somatic Somatic Somatic Somatic

1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 4 (6.1%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%)

0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 3 (0.8%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%)

Somatic Somatic Somatic Germline Somatic Germline Germline Somatic Somatic Somatic Somatic Somatic Somatic Germline Somatic Somatic Somatic

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Table S1. Cont. Position

Gene

Change

Amino-Acid Change or Watson-Crick Base-Pairing

14178 14310 14463 14518 14569 14587 14668 14774 14783 15018

ND6 ND6 ND6 ND6 ND6 ND6 ND6 Cytb Cytb Cytb

T–C C–A T–C A–G G–A A–G C–T C–A T–C T–A

No: Ile -> Val No: Gly -> Trp No: Thr -> Ala Gly -> Gly Ser -> Ser Gly -> Gly Met -> Met No: Leu -> Ile Leu -> Leu No: Phe -> Tyr

16108 16162 16164 16166 16172 16182 16183 16184 16185 16189 16223 16259 16304 16362 16390 16482 16519

D-Loop D-Loop D-Loop D-Loop D-Loop D-Loop D-Loop D-Loop D-Loop D-Loop D-Loop D-Loop D-Loop D-Loop D-Loop D-loop D-Loop

C–T A–G A–G A–G T–C A–C A–C C–T C–T T–C C–T C–T T–C T–C G–A A–G T–C

– – – – – – – – – – – – – – – – –

Conservation Reported b Index (%) a Protein-coding region 82.9% Y 70.7% N 90.2% Y 100% Y 100% Y 100% Y 36.6% Y 63.4% N 26.8% Y 100% N D-Loop region – Y – Y – Y – Y – Y – Y – Y – Y – Y – Y – Y – Y – Y – Y – Y – Y – Y

Number of 66 PTC Patients (%)

Number of 376 Healthy Controls (%)

Somatic/Germline

3 (4.5%) 1 (1.52%) 1 (1.52%) 2 (3.03%) 2 (3.03%) 4 (6.1%) 12 (18.18%) 1 (1.52%) 28 (42.42%) 1 (1.52%)

3 (0.8%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 14 (3.72%) 3 (0.8%) 59 (15.69%) 0 (0.00%) 165 (43.88%) 0 (0.00%)

Somatic Somatic Somatic Somatic/Germline Somatic/Germline Somatic/Germline Somatic/Germline Somatic Somatic/Germline Somatic

3 (4.5%) 3 (4.5%) 4 (6.1%) 1 (1.52%) 7 (10.6%) 8 (12.12%) 16 (24.24%) 2 (3.03%) 2 (3.03%) 21 (31.81%) 31 (46.96%) 2 (3.03%) 7 (10.6%) 3 (4.5%) 1 (1.52%) 1 (1.52%) 3 (4.5%)

2 (0.53%) 12 (3.19%) 6 (1.6%) 2 (0.53%) 34 (9.04%) 38 (10.11%) 87 (23.14%) 6 (1.6%) 10 (2.66%) 112 (29.79%) 183 (48.67%) 1 (0.27%) 47 (12.5%) 114 (30.32%) 3 (0.8%) 0 (0.00%) 146 (38.83%)

Somatic/Germline Somatic/Germline Somatic/Germline Somatic Somatic/Germline Somatic/Germline Somatic/Germline Somatic Somatic Somatic/Germline Somatic/Germline Somatic Somatic/Germline Somatic/Germline Somatic Somatic Germline

Conservation index denotes the conservative properties of amino-acid or nucleotides in 41 primate species; c Ter: Terminator. a

b

According to Mitomap (http://www.mitomap.org);

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Table S2. Potentially pathogenic mtDNA variations in RNA region. Position

Gene

Change

654 2274 3183

12S rRNA 16S rRNA 16S rRNA

T–C A–G T–C

3244

tRNALeu(UUR)

G–A

3275–3276 4272 4417 5514 5628 5814 5835 5881 10463

tRNALeu(UUR) tRNAIle tRNAMet tRNATrp tRNAAla tRNACys tRNATyr tRNATyr tRNAArg

Del CA T–C A–G A–G T–C T–C Ins T G–C T–C

Watson-Crick Base-Pairing a G–C↑

A–U↓ A–U↓ A–U↓

C–G↓

Conservation Index (%) b 90.20% 100.00% 80.50%

Y N N

Number of 66 PTC Patients (%) 1 (1.52%) 1 (1.52%) 1 (1.52%)

Number of 16 Normal Thyroid Tissues (%) 0 (0.00%) 0 (0.00%) 0 (0.00%)

Number of 376 Healthy Controls (%) 0 (0.00%) 0 (0.00%) 0 (0.00%)

100.00%

Y

1 (1.52%)

0 (0.00%)

0 (0.00%)

MELAS, elderly brain,gastric carcinoma, lung tumor, thyroid oncocytoma

100.00% 97.60% 93.75% 87.50% 85.40% 100.00% 92.70%

N N Y Y Y Y N N Y

1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%) 1 (1.52%)

0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%)

0 (0.00%) 0 (0.00%) 0 (0.00%) 2 (0.53%) 1 (0.27%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%)

Neonatal onset mito disease CPEO/DEAF enhancer Mitochondrial Encephalopathy

Reported c

Disease-Associated Mutations Reported in Mitomap

Endometrium tumor

Watson-Crick base-pairing: created (↑) or abolished (↓); b Conservation index denotes the conservative properties of amino-acid or nucleotides in 41 primate species; c According to Mitomap (http://www.mitomap.org). a

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Table S3. Clinicopathological significance of pathogenic mtDNA mutations in the protein coding region.

Pathogenic mtDNA Mutation Yes No p Value Age (year) 45 22 13 Sex Male 8 5 0.321 Female 24 28 Micro-PTC a No 28 24 0.137 Yes 4 9 Bilaterality Unilateral 20 19 0.685 Bilateral 12 14 Multifocality Single 18 16 0.531 Multiple 14 17 With Hashimoto thyroiditis b No 27 28 0.796 Yes 4 5 Adenoma b No 30 30 0.333 Yes 1 3 Extrathyroid invasion No 27 30 0.423 Yes 5 3 Lymph node metastasis No 19 18 0.694 Yes 13 15 Tumor stage I–II 19 28 0.022 * III–IV 13 5 Recurrence b No 20 18 0.508 Yes 5 7 Novel mtDNA variation No 11 26 Leu Gly -> Gly Gly -> Gly

Conservation Index (%) b

58.50% 4.90% 85.40% 97.60% 100.00% 100.00%

PTC Group (n = 66) 4 11 4 10 35 10 65 30 7 5 6 6 4 4 30

Control Group (n = 376) 6 22 6 114 146 18 303 116 7 67 11 9 6 5 91

OR (95% CI) 3.98 (1.091 to 14.51) 3.22 (1.479 to 7.004) 3.98 (1.091 to 14.51) 0.41 (0.2022 to 0.8331) 1.78 (1.051 to 3.010) 3.55 (1.560 to 8.088) 15.66 (2.137 to 114.8) 1.87 (1.097 to 3.179) 6.25 (2.117 to 18.48) 0.38 (0.1463 to 0.9769) 3.32 (1.183 to 9.311) 4.08 (1.401 to 11.87) 3.98 (1.091 to 14.51) 4.79 (1.251 to 18.32) 2.61 (1.522 to 4.474)

Frequency in 16 Normal Thyroid Tissues (%) 0.00% 12.50% 0.00% 43.75% 43.75% 12.50% 87.50% 43.75% 6.25% 31.25% 0.00% 0.00% 0.00% 0.00% 43.75%

Watson-Crick base-pairing: abolished (↓); b Conservation index denotes the conservative properties of amino-acid or nucleotides in 41 primate species; The mtSNPs had the influence in the predisposition of thyroid cancer were highlighted by bold and italic.

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Table S6. mtDNA sequence of 41 vertebrate species.

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Species Name Cebus albifrons Chlorocebus aethiops Chlorocebus pygerythrus Chlorocebus sabaeus Chlorocebus tantalus Colobus guereza Daubentonia madagascariensis Eulemur fulvus fulvus Eulemur fulvus mayottensis Eulemur macaco macaco Eulemur mongoz Galago senegalensis Gorilla gorilla Gorilla gorilla gorilla Homo sapiens Homo sapiens neanderthalensis Hylobates lar Lemur catta Loris tardigradus Macaca mulatta Macaca sylvanus Nasalis larvatus Nycticebus coucang Otolemur crassicaudatus Pan paniscus Pan troglodytes Papio hamadryas Perodicticus potto Piliocolobus badius Pongo abelii Pongo pygmaeus Presbytis melalophos Propithecus coquereli Pygathrix nemaeus Rhinopithecus roxellana Saimiri sciureus Semnopithecus entellus Tarsius bancanus Tarsius syrichta Trachypithecus obscurus Varecia variegata variegata

Genbank Accession Number NC_002763.1 NC_007009.1 NC_009747.1 NC_008066.1 NC_009748.1 NC_006901.1 NC_010299.1 NC_012766.1 NC_012769.1 NC_012771.1 NC_010300.1 NC_012761.1 NC_001645.1 NC_011120.1 NC_012920.1 NC_011137.1 NC_002082.1 NC_004025.1 NC_012763.1 NC_005943.1 NC_002764.1 NC_008216.1 NC_002765.1 NC_012762.1 NC_001644.1 NC_001643.1 NC_001992.1 NC_012764.1 NC_008219.1 NC_002083.1 NC_001646.1 NC_008217.1 NC_011053.1 NC_008220.1 NC_008218.1 NC_012775.1 NC_008215.1 NC_002811.1 NC_012774.1 NC_006900.1 NC_012773.1

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Figure S1. Distribution of mtDNA variations in PTC. (a) Distribution of mtDNA variations in the entire mitochondrial genome; (b) Variation load in genes and complexes; (c) Distribution of different types of variation; (d) The ratio of synonymous and nonsynonymous variations in different types of substitution; (e) The ratio of synonymous and nonsynonymous variations in genes of protein-coding region; (f) Variation load of synonymous and nonsynonymous variations in genes and complexes.