Mitochondrial Heteroplasmy

Transcription

1 Mitochondrial Heteroplasmy Sabine Lutz-Bonengel Institute of Legal Medicine, University Hospital of Freiburg, Freiburg, Germany

2 Outline Characterization of length and sequence heteroplasmy Heteroplasmic sites Problems Heteroplasmy in single cells and triplets

3 Definition Mitochondrial heteroplasmy: presence of a mixture of more than one type of mtdna within an individual

4 Differentiation of length and sequence heteroplasmy length heteroplasmy sequence heteroplasmy

5 Length heteroplasmy Variants differ in the number of bases Elevated sequence background C11/12

6 Sequence heteroplasmy Variants differ in the type of bases Superimposition of usually two different bases at one position 195Y 234R

7 Length heteroplasmy - Mechanism PCR + sequencing C7TC6 replication slippage >10% PCR + sequencing C7/8TC6 C7TC6 C8TC6

8 Sequence heteroplasmy - Mechanism PCR + sequencing Point mutation >20% PCR + sequencing A, C, G, T

9 Length heteroplasmy - Sites C5TC4 C7TC5 (AC) 5 C6

10 Positions C5TC4 according to rcrs Bendall & Sykes 1995, Am J Hum Genet 57, 248ff

11 according to rcrs AAAACCCCCTCCCCA T16189C AAAACCCCCCCCCCCN T16189Y A16183C; T16189C AAAACCCCCYCCCCA AAACCCCCCCCCCCN A16182C; A16183C; T16189C AACCCCCCCCCCCCN

12 according to rcrs CCCCCTCCCC T16189C CCCCCCCCCCCN C16186T; T16189C CCTCCCCCCC C16188T; T16189C CCCCTCCCCCC T16189C; C16193T CCCCCCCCTC

13 Length heteroplasmy - Sites C5TC4 C7TC5 (AC) 5 C6

14 Positions C7TC6 according to rcrs Greenberg et al. 1983, Gene 21, 33ff

15 Length heteroplasmy: according to rcrs

16 Length heteroplasmy: C according to rcrs

17 Length heteroplasmy: C according to rcrs 309.2C

18 Length heteroplasmy: according to rcrs T310C

19 Length heteroplasmy - Sites C5TC4 C7TC5 (AC) 5 C6

20 Positions (AC) 5 according to rcrs

21 Length heteroplasmy: AC-repeat 523 del; 524 del according to rcrs A; C A; C A; C

22 Sequence heteroplasmy - Sites C5TC4 C7TC5 (AC) 5 C6

23 Positions (C) 6 according to rcrs

24 Length heteroplasmy: according to rcrs C; C? C; C C;?

25 Sequence heteroplasmy - Sites T C A CC C C T T A G C T C GA T C T TC G C A T C T C

26 Sequence heteroplasmy - Sites 195Y 16291Y 234R 152Y

27 Problems

28 Detection of length heteroplasmy Blurred sequence following length heteroplasmy

29 Detection of length heteroplasmy Blurred sequence preceding length heteroplasmy

30 Detection of length heteroplasmy When do we have to report heteroplasmy? homoplasmic sample C7TC6 weak heteroplasmic sample C8TC6 C-insertion at pos strong heteroplasmic sample C8/9TC6 C-insertion(s) at pos /.2

31 Detection of length heteroplasmy Phantom mutations due to software Brandstätter et al. 2005, Electrophoresis 26, 3414ff

32 Sequencing downstream length heteroplasmy Use of internal / junction primers Szibor & Michael 1999, Int J Legal Med 112, 348ff / Rasmussen et al. 2002, For Sci Int 129, 209ff

33 Sequencing downstream length heteroplasmy double reactions from the same strand but from two PCR products F15 F29 PCR 1 PCR 2 R484 R639 Parson & Bandelt 2007, For Sci Int Gen 1,13ff

34 Detection of sequence heteroplasmy with high sequence background high sequence background Brandstätter & Parson 2003, Int J Legal Med 117, 180ff

35 Detection of sequence heteroplasmy using different primers primer L29 L16450 proportion ~ 2:1 ~ 6:1 position 234R Brandstätter & Parson 2003, Int J Legal Med 117, 180ff

36 Differentiation of heteroplasmy from mixed stains or contaminations Y Y Y Y Separation by cloning T T T Confirmation by STRanalysis 1) 2) C Compare to control samples

37 Differentiation of heteroplasmy from polymerase errors Analysis of two PCR products

38 Heteroplasmy in different tissues of an individual tissue buccal cells blood A/G A/g proportion 1:1 4:1 position 234R

39 Heteroplasmy in different hairs of an individual tissue hair 1 hair 2 hair 3 G G/a A/g proportion 15:1 3:1 ~1:2 position 234R

40 Heteroplasmy in the course of life Lagerström-Fermér et al. 2001, Am J Hum Genet 68, 1299ff Del Bo et al. 2002, J Neurol Sci 202, 85ff

41 Sequence heteroplasmy in the maternal pedigree T C/t Grandmother Mother position 204Y C/t T/C T Daugther 1 Son 1 Daugther 2 Lo et al. 2005, Ann NY Acad Sci 1042, 130ff

42 Length heteroplasmy in the maternal pedigree number of C-residues between positions : Lutz et al. (2000) Int J Legal Med 113, 155ff

43 Length heteroplasmy in the maternal pedigree number of C-residues between positions : Lutz et al. (2000) Int J Legal Med 113, 155ff

44 Length heteroplasmy in triplets Monozygous C8 C9 Sibling 1 Nonmonozygous Nonmonozygous C8 C9 Sibling 2 C8 C9 Sibling 3 RFLP-Analysis Lutz-Bonengel et al. (2004) Int J Legal Med 118, 274ff

45 Length heteroplasmy in triplets Triplets "Distance" Monozygous siblings Non-monozygous siblings

46 Level of heteroplasmy? Tissue Cell Mitochondria

47 Sequence heteroplasmy within single cells Results of single cells PCR sequencing Single cells minisequencing Lutz-Bonengel et al. Int. J. Legal Med. (in press)

48 Level of sequence heteroplasmy Tissue 96% Cell 4%? Mitochondria Lutz-Bonengel et al. Int. J. Legal Med. (submitted)

49 Length heteroplasmy within single cells Results of single cells PCR sequencing Single cells RFLP C7C8 C8C9

50 Level of length heteroplasmy Tissue Cell Mitochondria

51 Conclusions Agreement: when and how do we have to report heteroplasmy? Pitfalls: be aware of possible errors of interpretation Detection depends on quality and sensitivity of the used method No exclusions in casework because of heteroplasmic sites Increased discrimination power by corresponding results

52 Thank you for your attention! Questions? Comments? Volunteers?