Pit fall s with mtDNA analysis in medical genetics Hans-Jürgen Bandelt (Hamburg)

1. Pitfalls with mtDNA analysis in medical geneticsHans-Jürgen Bandelt (Hamburg) EMBO World Programme Workshop on Human Evolution and Disease, Hyderabad, India. 6th – 9th December 2006

2. From the journal Oncogene, this year, one could pick up the following exciting news: “Very strikingly, the mitochondrial sequences in this study (tumor samples as well as controls) with the Indian population revealed a unique profile of eight sequence variants, viz. A73G, A263G, A1438G, A2706G, A4769G, C7028T, A8860G and A15326G appeared at high frequencies in all samples and could be of evolutionary significance.”

3. “... the profile could be specific to theIndian population.” R0 = Well, not, quite ...

4. Leaving aside mutations that weresystematically missed (such as A750G and C14766T, probably due to the use of a wrong reference sequence), the claim would translate into: ... Near-absence of haplogroup R0 could be specific to the Indian population. Note that haplogroup R0 is specific to the West Eurasian mtDNA pool.

5. Absence of phylogenetic knowledge about human mitochondrial DNA is characteristic of clinical genetics.

6. In a paper from Muscle and Nerve, 2003, the authors (from Taipei) identified among completely sequenced 17 cases “a double mutation (A3243G and A14693G) in a patient with MELAS syndrome, who had a diabetic mother and normal siblings. The A14693G substitution is significant from structural and evolutionary points of view. This result indicates that mtDNA should be sequenced in its entirety for the complete evaluation of mitochondriopathy.” However, the complete mtDNA sequence of that MELAS patient was not reported ...

7. “A14693G is not identified in 205 human controls and 76 randomly examined species.” Y1b Y2 15460 15221 10097 146 Y1a 16311 15244 14914 7859 6941 5147 482 7933 Y1 However, it has been known since 2003 that A14693G is a characteristic mutation of the East Asian haplogroup Y 16266 3834 Y 16231 16223 16126 14693 14178 10398 8392 N9 5417 N Bandelt, Yao & Kivisild (2005)

8. According to Trejaut et al. (2005), haplogroup Y2 does occur in parts of Taiwan

9. Suppose that you would now find A14693G in some disease context and would consult MITOMAP prior to publishing your case:

11. Green light from MITOMAP, thus – and here you go: Dong-Ling Tang, Xin Zhou, Xia Li, Lei Zhao, Fang Liu Diabetes Res Clin Pract. 2006 Jan 13 “… in a Chinese population, a total of 184 T2DM cases and 279 matched healthy controls were recruited. … Our results suggest that the mutations of T3394C and A14693G may contribute to genetic predisposition to T2DM, with the T16189C variant being associated with insulin resistance.” Dong-ling Tang, Xin Zhou, Ke-yuan Zhou, Xia Li, Lei Zhao, Fang Liu, Fang Zheng, Song-mei Liu Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2005 Dec 22 “A total of 184 cases of type 2 diabetes mellitus and 210 matched healthy controls with normal glucose tolerance were recruited for the study. …. The mutations of 3394 (T-->C) and 14693 (A-->G) may contribute to the genetic predisposition to type 2 diabetes; 16189 (T-->C) variant is associated with insulin resistance and risk factor of diabetes.”

12. Double-publication does not seem to be a rare phenomenon in China. Another way of re-cycling is to publish one and the same mutation as novel multiple times – either found in one and the same patient who was re-examined again and again, or in several different patients – as long as the mutation would not find its way into MITOMAP.

13. The control group investigated by Tang et al. (2006) has the peculiar feature that in a sample of size 279 there was absolutely no haplogroup M9a‘b lineage (T3394C). Other studies would, however, rather suggest a figure of 2.7% (in Han Chinese). Under the hypothesis that this percentage is the true frequency, then the event of observing 0/279 M9a‘b lineages would have a probability of 0.05%!

14. There are further cases, in cancer research, where control group data are almost void of any variation and thus are either cooked or fabricated, e.g. taken over (via copy-and-paste) from other studies (which have a most peculiar error spectrum themselves).

15. Patients Controls Cooked data 2 3 4 5 1 CRS UC-Case 1 Control 1 Control 2 UC-Case 2 UC-Case 3 14766 14368G 14365G 14272G 14199G 13702G 11335 9559G 4985 3423G 3106del 16147 14570G 11956 11712G 11457G 11247G 11853 9883G 9860G 5942 4662 3338G 3285G 1486G 1000A 13586 12417 11853 11771G 11102+G 10999G 7337 500del 11955C 11947C 10590G 3969 2472del 16524+G 10027+G 9572 8494T 8483T 8021+A 7917+G 7904+G 6456 5101 4919+T 4032-4033del 2036+C 1080+C 1074+C 8850 8639 6479+A 2687del 741 5 F1a’c 16129 13759 9053 1 5 1 7852 rCRS False mutations M7a1a5 15326 8860 315+C 263 1 4 3 5 F1 2 1 4 3 5 522-523del 13768 1 2 4 3 5 12882 12406 10609 6962 1 D4c1a 2 3 1 4 5 B4 M7a1a 1 H2a 16223 9755 3391 207 199 194 191+A 1 11084 11017 750 3 2 1 5 4 3 1 16217 2 4 3 H2 M8a1 F 3 M7a1 4769 1438 3 2 5 3 16184 2835 2 4 1 5 3 16324 143645899+C 10310 6392 249del 4 B 1 5 H 3 7028 2706 D4c1 3 2 1 5 4 M8a 3 2 1 5 4 M7a 8281-8289del 16189 2 2766 3 HV 16319 14470 8684 6179 2 D4c 16209 12771 4958 4386 2772 2626 5 R9 14766 4 3 2 1 4 16245 R0=pre-HV 16304 13928C 3970 1 D4 1 11719 73 5 3 2 1 4 14668 8414 3010 3 4 M8 1 2 1 5 4 3 3 4 16298 15487T 8584 7196A 4715 R M7 D 5 16223 12705 5 4 5 16362 5178A 4883 3 9824 6455 4 5 3 3 Missed mutations 5 N M 15301 10873 10398 9540 8701 3 5 4 4 3 15043 14783 10400 489 3 4 3 3 4 3 3 4 5 4 5 3 4 3 5 L3

16. In particular, mtDNA analysis in cancer studies is riddled with all kinds of error – for example, sample mix-up or contamination: Bundles of perceived somatic mutations then actually trace parts of phylogenetic pathways in the mtDNA phylogeny (Salas et al. 2005).

17. How would authors of inflicted papers react? Well, they could say that the revisited pathway would just express the disease... In one remarkable case it was proclaimed: “This patient had a germline mitochondrial haplotype J, which “shifted” in positions 185, 295, and 16126 back to the phylogenetically older haplotype H, but shifted in position 195 to haplotype W and in position 204 to nowhere.”

18. Pythonesque...* *Said of a style of humour: bizarre and surreal

19. Monty Python is the collective name of the creators of Monty Python's Flying Circus, a British television comedy sketch show broadcast by the BBC from 1969 to 1974. MP‘s foot The Dead Parrot sketch is one of the most famous in the history of television comedy. Information and links: Wikipedia

20. The Dead Parrot sketch portrays a conflict between a disgruntled customer and a shopkeeper, who hold contradictory positions on the vital state of a Norwegian Blue parrot (an apparent absurdity in itself). "I know a dead parrot when I see one, and I'm lookin' at one right now."

21. The customer complains that the parrot he has recently purchased at the location is, in fact, dead. The shopkeeper denies this and points out the beauty of its plumage, further suggesting that the bird is merely asleep.

22. Monty Python's Dead Parrot sketch has come to life in molecular anthropology: The Caucasian King Size parrot starring Ivani Nasidze & Mark Stoneking from the MP Institute EVA

23. Natal history of the Caucasian King Size parrot: 2001 Nasidze and Stoneking published a Caucasian HVS-I data set (but did not show the data) 2003 At the International Symposium on Forensic DNA Technologies in Münster this Caucasian data set was accused of having an enormous (king size) error spectrum 2004 Nasidze and colleagues gave a false statement (in the Annals of Human Genetics) about the vital state of their old data set,by employing a sort of a filter analysis (however, non-adjusted to actual sequencing range):

24. “... (analysis not shown). These reticulations were just due to one sequence*, as removal of this sequence removed the excess reticulations.” Labels 16000+ False statement! Here‘s the torso of the reticulate network after removal of that sequence (Bandelt and Kivisild 2006). * Corrected Sequence not shown

25. Advertisement This Caucasian HVS-I data set has now been displayed publicly and can be inspected in the book: Human Mitochondrial DNA and the Evolution of Homo sapiens Bandelt, Hans-Jürgen; Richards, Martin; Macaulay, Vincent (Eds.) Springer-Verlag, 2006, 117.65€

28. Those who wish to evaluate the idiosyncratic variation of this Caucasian data set but have no prior knowledge of natural mtDNA variation may proceed as follows:

29. Take out all mutations ever observed in any one of the complete sequences referred to in Max Ingman‘s database (http://www.genpat.uu.se/mtDB/) and represent the surviving variation in the form of a quasi-median network that highlights the character conflicts and compare with other data sets:

30. Beautiful plumage From: Bandelt & Dür (2006)*

31. *Hans-Jürgen Bandelt & Arne Dür (2007) Translating DNA data tables into quasi-median networks for parsimony analysis and error detection. Molecular Phylogenetics and Evolution 42: 256–271. In this article it is demonstrated that the mtDB2005-filtered variation of the Caucasian data is even messier than corresponding randomised data tables.

32. The stone dead Caucasian King Size parrot: Bandelt & Kivisild (2006) Quality assessment of DNA sequence data: autopsy of a mis-sequenced mtDNA population sample. Annals of Human Genetics 70: 314–326. Stoneking & Nasidze (2006) The patient is not dead yet: premature autopsy of a mtDNA data set. Annals of Human Genetics 70: 327–331. Parson (2006) The art of reading sequence electropherograms. Annals of Human Genetics Stoneking & Nasidze (2006) Reply to Parson. Annals of Human Genetics

34. Remarkable electropherogram! What‘s wrong with it?

35. This electropherogram don't enter into it. That‘s what‘s wrong with it. No one has ever claimed that the transitionC16168T is a phantom mutation – instead, it‘s the transversionC16168A that may be a phantom mutation. In Nasidze et al. (2001) the 16168transition always occurs together with the 16343 transition: this constitutes a confirmed motif within haplogroup U3 (Macaulay et al. 1999).

36. Eight of these paired electropherograms are irrelevant here because they just show other sequences with the rCRS nucleotide in question. Instead, the heavy strand electropherograms could have been shown in these places!

37. Walther Parson has aptly demonstrated that those electropherograms reflect well-known and reproducible sequencer artifacts and were not interpretedproperly by Nasidze and Stoneking. They have always asserted that they have read both strands – butwithout ever giving any evidence for that: “As stated both in the original papers (Nasidze & Stoneking 2001: p.1198; Nasidze et al. 2004: p.207) and in the reply to Bandelt & Kivisild (Stoneking & Nasidze, 2006: p.329), both strands were indeed sequenced in all samples.” Thus, they are iterating a false statement.

38. You know a dead parrot when you see one: ... “(data not shown)”* * and not submitted to GenBank either ... “(analysis not shown)”

39. In case you wish to register a complaint, please, address yourself to the ombudsperson: “Scientific honesty and the observance of the principle of good scientific practice are essential in all scientific work which seeks to expand our knowledge and which is intended to earn respect from the public.”

40. From Mark Stoneking’s website (http://email.eva.mpg.de/~stonekg/files/ombud.htm): “As elected Ombudsperson of the Max Planck Institute for Evolutionary Anthropology, I stand at your disposal in case you are experiencing or observing any kind of scientific misconduct, or if you need advice on the subject of good scientific practice.” “Scientific misconduct includes: false statements, infringement of intellectual property, impairment of the research work of others, joint accountability.”

41. Acknowledgement: Sincere thanks are due to Martin Richards (reminding me of MP‘s immortal Dead Parrot sketch) and all collaborators (Anita Brandstätter, Claudio Bravi, Mike Coble, Arne Dür, Toomas Kivisild, Jüri Parik, Walther Parson, Antonio Salas, Richard Villems, Yong-Gang Yao)

42. Thank you for listening