1471-2164-7-185 1471-2164 Research article <p>The pseudo-mitochondrial genome influences mistakes in heteroplasmy interpretation</p> Parr L Ryan Ryan.Parr@genesisgenomics.com Maki Jennifer Jennifer.Maki@genesisgenomics.com Reguly Brian Brian.Reguly@genesisgenomics.com Dakubo D Gabriel Gabriel.Dakubo@genesisgenomics.com Aguirre Andrea Andrea.Aguirre@genesisgenomics.com Wittock Roy Roy.Wittock@genesisgenomics.com Robinson Kerry Kerry.Robinson@genesisgenomics.com Jakupciak P John john.jakupciak@nist.gov Thayer E Robert Robert.Thayer@genesisgenomics.com

Genesis Genomics Inc, 1294 Balmoral Street, Thunder Bay, Ontario, P7B 5Z5, Canada

Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA

 BMC Genomics 1471-2164 2006 7 1 185 http://www.biomedcentral.com/1471-2164/7/185 16859552 10.1186/1471-2164-7-185 19 4 2006 21 7 2006 21 7 2006 2006 Parr et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Nuclear mitochondrial pseudogenes (numts) are a potential source of contamination during mitochondrial DNA PCR amplification. This possibility warrants careful experimental design and cautious interpretation of heteroplasmic results.

Results

Here we report the cloning and sequencing of numts loci, amplified from human tissue and rho-zero (ρ0) cells (control) with primers known to amplify the mitochondrial genome. This paper is the first to fully sequence 46 paralogous nuclear DNA fragments that represent the entire mitochondrial genome. This is a surprisingly small number due primarily to the primer sets used in this study, because prior to this, BLAST searches have suggested that nuclear DNA harbors between 400 to 1,500 paralogous mitochondrial DNA fragments. Our results indicate that multiple numts were amplified simultaneously with the mitochondrial genome and increased the load of pseudogene signal in PCR reactions. Further, the entire mitochondrial genome was represented by multiple copies of paralogous nuclear sequences.

Conclusion

These findings suggest that mitochondrial genome disease-associated biomarkers must be rigorously authenticated to preclude any affiliation with paralogous nuclear pseudogenes. Importantly, the common perception that mitochondrial template "swamps" numts loci precluding detectable amplification, depends on the region of the mitochondrial genome targeted by the PCR reaction and the number of pseudogene loci that may co-amplify. Cloning and relevant sequencing data will facilitate the correct interpretation. This is the first complete, wet-lab characterization of numts that represent the entire mitochondrial genome.

Background

The unique maternal inheritance pattern of mitochondrial DNA (mtDNA), its small genome size, accelerated mutation rate, lack of recombination, and multiple copy number per cell, in comparison to nuclear DNA, are ideal biological traits for investigating evolution, population genetics and for forensic and medical applications. Thus, the mitochondrial genome has been used as a biosensor for the timing and movement of human populations in antiquity 12. MtDNA analysis is routinely used in forensic biology to type biological material when degradation prevents nuclear STR amplification 3. In addition, the entire mitochondrial molecule has potential medical utility because it can serve as a repository of cancer mutations and as a biosensor indicative of genetic alterations 45678910111213.

Frequently, identifying legitimate mtDNA mutations is confounded by heteroplasmy, a condition in which wild-type and mutant mitochondrial genomes co-exist in a cell. The interpretation of heteroplasmy can further be confounded by the widespread integration of portions of the mitochondrial genome into the nuclear genome 1415. These homologous, yet divergent nuclear and mtDNA sequences can be co-amplified in PCR reactions intended to replicate targeted mtDNA sequences only. Although this problem has previously been considered to be muted because of the high copy number of mtDNA over corresponding nuclear loci, caution is warranted 16. For example, there are specific regions of the mitochondrial genome that have corresponding nuclear mitochondrial pseudogenes (numts) distributed across multiple chromosomes. Hence, there are regions of the mitochondrial genome that have a high nuclear copy number, which are not completely "swamped" during amplification. We report that some heteroplasmies detected in prostate cancer samples are a result of co-amplification of these multiple loci.

A large number of manuscripts addressing errors related to the interpretation of mtDNA and mtDNA heteroplasmy has been published 171819202122232425. Notably not all these errors are due to pseudogene co-amplification; however, mistakes from pseudogenes may increase with improved sequencing methods and highly sensitive re-sequencing microarray technologies that have a lower detection limit than traditional sequencing and which readily detect low-level heteroplasmy 1126. In some cases, if the heteroplasmy is inherited, it substantially increases the power of mutation detection, which becomes an important aspect since heteroplasmy has been reported as an early indication of disease 2728293031. In addition, if the disease process invokes mitochondrial depletion, this could increase nuclear pseudogene signal in reactions as a result of reduced mitochondrial genome copy number 32. Loss of mitochondria has been described in several human cancers 33343536. As well, the number of mitochondria and mtDNA copy number vary for different cell types 373839. These important matters relating to sequence interpretation have been generally neglected, in part, due to the lack of numt reference material, which would help investigators determine the relevance of detected mtDNA sequence variations. Hence, the need to validate somatic mitochondrial mutations is a pressing one.

Heteroplasmic issues have already complicated data obtained from other species. For example, in elephant hair, low mtDNA content is the reason why numts were co-amplified and misinterpreted as authentic mtDNA. In contrast, numts were not detected in DNA derived from elephant blood due to the presence of mitochondria-rich platelets 40. Moreover, the hominid, Gorilla, is well known for significant numt interference with mitochondrial sequences, highlighting the need for diligence when interpreting human mtDNA heteroplasmy 41. Not surprisingly, the effort for using mitochondrial cytochrome c oxidase as a primate "barcode" is plagued by numt amplification as well 42.

Further, laser capture microscopy has improved the ability to separate and analyze cancer cells, but because of the decreased amounts of sample DNA, many primer pairs are required to obtain a robust amplification of the entire mitochondrial genome 43. Moreover, a sufficient number of cells must be captured to avoid incorporation errors associated with low template quantity 44. This will also be relevant to studies that use formalin-fixed paraffin embedded samples 45. The use of many primers means that smaller amplicons will be synthesized translating into a higher risk of co-amplification of numts and the potential misinterpretation of heteroplasmic calls.

There is limited in silico and wet-lab evidence indicating that fragments of the human mitochondrial genome are embedded in the nuclear DNA archive 4647484950. These findings emphasize the critical need to minimize erroneous interpretation of heteroplasmy, a vital necessity for precise forensic discrimination, evolutionary studies, and potential diagnostics. We provide evidence of numts for the entire mitochondrial genome by the amplification, cloning, and identification of numts from rho-zero (ρ0) cells and clinical cancer specimens. Here we present results from overlapping primers, which co-amplified nuclear embedded, paralogous mitochondrial sequence. Surprisingly, our data shows a relatively small number (when compared to hypothetical sequence information obtained from BLAST searches) of multiple nuclear loci that co-amplify with the mitochondrial genome. These findings demonstrate that accurate interpretation of heteroplasmy not only requires careful primer design and testing, but also indicates that a compendium of the sequence information from multiple-copy number numts is an important reference tool that will facilitate correct mtDNA interpretation and support reliable mitochondrial genome sequencing data.

Results

ρ0 cells lack mtDNA

Rho-zero cells were evaluated for the presence of mtDNA. To ensure that total DNA extracted from ρ0 cells were indeed devoid of mtDNA, we first performed Southern blot analyses on DNA extracts from ρ0 cells. Blood was used as a positive control. No full length mitochondrial genome signal was observed in ρ0 lanes when the blots were probed with mtDNA-specific probes (Figure 1a). We next performed PCR on the DNA extracts with primers specific to the mitochondrial coding regions. Again, there were no amplifications observed in the ρ0 templates, while DNA isolated from blood was amplified, as expected (data not shown). We used RT-PCR to provide further evidence that ρ0 cells are indeed devoid of mtDNA 51. RT-PCR analysis was performed on RNA samples from ρ0 cells and normal human skin tissue (epithelial cells) samples with primers to OXPHOS genes and a nuclear gene (positive control), 5-aminolevulinate synthase (hALAS) (Table 1). Whereas the hALAS primers amplified nuclear targets in template from ρ0 and epithelial cells, there was no observable product with mtDNA primers for the ρ0 cDNA template (Figure 1b), independently confirming the absence of mtDNA in these cells.

<p>Figure 1</p>

ρ0 cellscells do not contain mtDNA

ρ0 cells do not contain mtDNA. a. Southern blot analysis of total DNA extracted from blood (bld) and ρ0 cells and probed with a full length mtDNA probe. Note the absence of hybridization to ρ0 extracts. Lad is a DIG-labeled DNA molecular weight marker III (Roche). b. PCR amplification of cDNA from ρ0 and epithelial cells (EC). Note the amplification of ρ0 cDNA with primers to the nuclear gene hALAS, whereas primers to ND1, ATPase6, and CYTB failed to amplify ρ0 cDNA, although they all amplified cDNA from EC. Lad is a 100bp DNA size standard (Fermentas life sciences).

<p>Table 1</p>

Sequences of four primer sets used in RT-PCR.

Designation

Primer sequence (5'-3')

hALASF

CCACTGGAAGAGCTGTGTGA

hALASR

ACCCTCCAACACAACCAAAG

ND1F

GAGCAGTAGCCCAAACAATC

ND1R

GGGTTCGGTTGGTCTCTGCTAG

ATP6F

CCATAAAATTATGAGCGGGCACAGTGATT

ATP6R

GGAAGGTTAATGGTTGATATTGCTAGG

CYTBF

CTAGCAACACTCCACCTCCTAT

CYTBR

GTAAGCCGAGGGCGTCTTTGCTTG

Co-amplification of numts and mtDNA

Amplification of the complete mitochondrial genome was performed on human formalin fixed and paraffin embedded (FFPE) prostate cancer samples using a set of 34 primers (Table 2). Due to the amount and quality of DNA recovered, the average amplicon size was limited to 625 bp. Surprisingly, 24 (71%) of the primer sets co-amplified mitochondrial pseudogenes (Figure 2, and data not shown). A similar ratio was previously reported by an independent group using 38 primers (26/38 or 68%) 16. In an effort to fully characterize numts that represent the entire mitochondrial genome, we redesigned the remaining 10 primers to co-amplify nuclear loci. Thus, we amplified template from ρ0 cells and subsequently identified (via sequencing) the cloned fragments from the nucleus. A region of the D-loop (base pairs 16211-420 and 15-711) was recalcitrant to co-amplification using our mitochondrial primers. Therefore, two chromosome 17 specific primers were designed to capture this D-loop fragment (Table 2). Hence, a total of 36 primer sets were used to recover the entire mitochondrial genome from the nucleus. The sequences representative of the entire mitochondrial genome are provided as an additional material (additional file 1). Figure 3 is an example of an alignment to rCRS of three numt clones recovered using primer set 1488F/2084R (Table 2). These three clones were recovered form three separate chromosomes (Chr11 – NT_009237, Chr5 – NT_006713, and Chr3 – NT_005612). Similar alignment of our consensus cloned sequences enabled the assembly of a pseudo-mitochondrial genome (Figure 4).

<p>Figure 2</p>

Numts co-amplify from clinical samples

Numts co-amplify from clinical samples. A representative gel picture showing amplification of clinical samples with primers that also amplify ρ0 template is shown. Unlabelled lanes are the clinical samples. Subsequent analysis of sequences from this amplification (see Figure 5) revealed the presence of pseudogene contamination.

 <p>Figure 3</p>

Example of an alignment of three clones

Example of an alignment of three clones. Example of an alignment of three clones (clones G C11. A1, G C3. A1 and G C5.C1) recovered from three chromosomes (Chr11 - NT_009237, Chr5 - NT_006713, and Chr3 - NT_005612) to the rCRS is shown.

 <p>Additional File 1</p>

Sequences of numt clones. The chromosomal locations and sequences of numts amplified using our primers

Click here for file

The following chromosomes were represented in the data: 1, 2, 3, 4, 5, 7, 8, 9, 11, 16, 17, 20 and X. The number of paralogous sequences, in some instances, was lower than the number predicted from BLAST searches (Figure 3, 5). We demonstrate that there are only a limited number of multiple copy numts that potentially contribute to a heteroplasmic signal. Subsequently, we systematically inspected heteroplasmic sites observed in sequences from the prostate cancer samples for numt contribution using our cloned ρ0 data as a reference. We discovered false heteroplasmic sites occurred when there was co-amplification of multiple numt loci with the same nucleotide at that particular site. Base pairs 1709, 1711 and 1719 in one specific amplicon (1488–2084, 16S rRNA) illustrate this point. The amplification of this specific region of the mitochondrial genome also co-amplified numts on chromosomes 3, 5 and 11 (Figures 3, 6). All three chromosomes have an A as opposed to a G, which correspond to mitochondrial positions 1709 and 1719. Using automated DNA sequencing, these multi-copy numts were detected as heteroplasmies at positions 1709 and 1719 (Figure 6). At position 1711, chromosomes 5 and 11 have a C as does the tissue; however, chromosome 3 has a T. A weak heteroplasmic signal is evident by a minute T peak, but because of the poor detection limit of fluorescent sequencing, this peak is virtually equivalent to background (Figure 6). Heteroplasmic signals were detected for other sites as well. For instance, the primer pair for the amplicon (3230–3893) co-amplified homologous numts on 5 different chromosomes (2, 4, 16, 17 and X). This region is evident in the pseudo-mitochondrial genome assembly from our clones (Figure 4).

<p>Table 2</p>

5' genome location (according to rCRS) and primer sequences for 36 primers including 34 (#3–36) used to amplify formalin fixed, paraffin embedded tissue. Primers in bold (1, 2) indicate chromosome (Chr) 17 specific primers redesigned to capture rest of D-loop fragment. The region spanned by 15971–16410 was split into two separate amplicons for the ρ 0 amplification (15673–16009 and 15777–16398).

Primer Sequence (5'-3')

Mitochondria Position

Chr

Accession Number

Nuclear Position

1

16319F

ATC GCT CAT GGT AGA TAG CAC

16319-446

17

NT_024862.13

337395–338087

446R

TGT TAG TTG AGG GGT GAC TGTT

2

374F

AAT CCA ACT CAA CCA GAG CCC A

371–461

17

NT_024862.13

338011–338101

781R

TGA GCT GCA CTA ATG CGT GC

Nuclear Gap

17

NT_024862.13

338102–338279

574–783

17

NT_024862.13

338280–338489

3

615F

ATG TTT AGA CGG GCT CAC ATC ACC

651–1247

11

NT_009237.17

9318994-9318400

1247R

CAA GAG GTG GTG AGG TTG ATC G

4

1051F

ACA ATA GCT AAG ACC CAA ACT GGG AT

1062–1639

11

NT_009237.17

9318585-9318008

1644R

CTC CTA AGT GTA AGT TGG GTG CTT TG

1068–1618

5

NT_006713.14

30541827-30541277

5

1488F

CGT CAC CCT CCT CAA GTA TAC TTC

1497–2049

11

NT_009237.17

9318150-9317598

2084R

TAC AAG GGG ATT TAG AGG GTT CTG TG

1497–2049

5

NT_006713.14

30541398-30540846

1497–2049

3

NT_005612.14

2832399-2832340

6

1938F

AGA GCA CAC CCG TCT ATG TAG CAA

1938–2612

11

NT_009237.17

9317709-9317035

2612R

GGA ACA AGT GAT TAT GCT ACC TTT GCA C

1938–2612

3

NT_005612.14

2831958-2831284

1939–2612

17

NT_024862.13

339611-340281

1940–2612

8

NT_023678.15

902509-901844

1971–2612

5

NT_006713.14

30540924-30540284

7

2417F

CAC TGT CAA CCC AAC ACA GGC AT

2402–3078

20

NT_011362.9

20987219-20986564

3101R

TAG AAA CCG ACC TGG ATT ACT CCG

2417–3076

17

NT_024862.13

340090-340749

8

3021F

CGC TAT TAA AGG TTC GTT TGT TC

3070–3673

17

NT_024862.13

340743-341346

3695R

ATC AGG GCG TAG TTT GAG TTT G

9

3230F

GTT AAG ATG GCA GAG CCC GGT AA

3253–3859

4

NT_016354.17

80880160-80879559

3893R

GTT CGG TTG GTC TCT GCT AGT GT

3253–3870

2

NT_005058.14

9588111-9587524

3296–3870

16

NT_037887.4

3360917-3360343

3323–3870

X

NT_011630.14

2761822-2761281

3347–3870

17

NT_024862.13

341020-341543

10

3728F

CAT ATG AAG TCA CCC TAG CCA TC

3814–4391

17

NT_010718.15

19103293-19103865

4417R

TTT AGC TGA CCT TAC TTT AGG ATG GG

3816–4391

17

NT_024862.13

341489-342064

11

4337F

ATG AGA ATC GAA CCC ATC CCT GAG

4361–5010

1

NT_077913.2

43543-44192

5035R

CAT CCT ATG TGG GTA ATT GAG GAG T

12

4867F

GAC AAA AAC TAG CCC CCA TCT CAA

4866–5554

1

NT_077913.2

44048–44736

5646R

GCT TAA TTA AAG TGG CTG ATT TGC GT

5169–5632

2

NT_005403.15

62851661-62852120

13

5468F

CAC GCT ACT CCT ACC TAT CTC

5508–6124

17

NT_024862.13

343174–343794

6145R

CAG TTG CCA AAG CCT CCG ATT ATG

5511–6121

1

NT_077913.2

44693–45303

14

5867F

CAA TGC TTC ACT CAG CCA TTT TAC C

5892–6481

1

NT_077913.2

45074–45663

6482R

GAC TGC TGT GAT TAG GAC GG

15

6418F

AAC CCC CTG CCA TAA CCC AAT AC

6441–7056

1

NT_077913.2

45623-46239

7082R

GAA GCC TCC TAT GAT GGC AAA TAC AG

16

6911F

TGC AGT GCT CTG AGC CCT AGG ATT

6914–7521

1

NT_077913.2

46097-46704

7554R

CTT TGA CAA AGT TAT GAA ATG GTT TTT CTA ATA

6935–7521

5

NT_034772.5

1804907-1804321

17

7400F

CCC ACC CTA CCA CAC ATT CGA A

7453–8005

1

NT_077913.2

46636–47188

8029R

GGC TTC AAT CGG GAG TAC TAC TCG

18

7829F

CGC ATC CTT TAC ATA ACA GAC GAG G

7853–8472

1

NT_077913.2

47036–47622

8441R

GTT GGG TGA TGA GGA ATA GTG TAA GG

17

8346F

CAACACCTCTTTACAGTGAAATGCCC

8471–8941

1

NT_077913.2

47652–48122

8959R

CGATAATAACTAGTATGGGGAT

20

8814F

CCA ACT ATC TAT AAA CCT AGC C

8814–9427

1

NT_077913.2

47995–48594

9413R

GCC TTG GTA TGT GCT TTC TCG TGT

8816–9375

5

NT_034772.5

1803025-1802466

21

9247F

GCC CAT GAC CCC TAA CAG G

9268–9845

5

NT_034772.5

1802573-1801994

9868R

CGG ATG AAG CAG ATA GTG AGG

9545–9859

7

NT_079592.1

56744603-56744909

22

9711F

CTG GGT CTC TAT TTT ACC CTC C

9730–10300

5

NT_034772.5

1802112-1801539

10285R

GGT AGG GGT AAA AGG AGG GCA

23

10198F

CCC GCG TCC CTT TCT CCA T

10206–10768

5

NT_034772.5

1801633-1801071

10766R

TTA GCA TTG GAG TAG GTT TAG G

24

10696F

CCC TAC TAG TCT CAA TCT CCA A

10694–11509

9

NT_008470.17

2194293–2194999

11426R

CTT CGA CAT GGG CTT TAG GGA G

10751–11426

5

NT_034772.5

36678748-36678073

25

11210F

TTC TAC ACC CTA GTA GGC TCC CTT

11234–11786

5

NT_034772.5

36678265-36677713

11813R

GTA GAG TTT GAA GTC CTT GAG AGA GG

26

11629F

AAT CAG CCA CAT AGC CCT CGT AG

11651–12205

5

NT_034772.5

36677848-36677294

12231R

GTT AGC AGT TCT TGT GAG CTT TCT CG

11841–12206

5

NT_034772.5

1799998-1799633

27

12096F

TCC TAT CCC TCA ACC CCG ACA T

12118–12681

5

NT_034772.5

36677381-36676818

12709R

GGA AGA TGA GTA GAT ATT TGA AGA ACT G

28

12528F

GAA CTG ACA CTG AGC CAC AAC C

12550–13070

5

NT_034772.5

1799289-1798769

13096R

CAA CTA TAG TGC TTG AGT GGA GTA GG

29

12882F

CAT CCT CGC CTT AGC ATG ATT TAT CC

12908–13493

5

NT_034772.5

36676591-36676006

13516R

GGT CTT TGG AGT AGA AAC CTG TG

12908–13493

5

NT_034772.5

1798931-1798346

30

13239F

CGT AGC CTT CTC CAC TTC AAG TC

13262–13827

5

NT_023148.12

2217633-2218198

13851R

GTT GAG GTC TAG GGC TGT TAG AAG

31

13354F

TTT ATG TGC TCC GGG TCC ATC AT

13377–13935

5

NT_023148.12

2217748-2218306

13957R

CTA GAT AGG GGA TTG TGC GGT G

32

13838F

CCC TAG ACC TCA ACT ACC TAA CC

13895–14454

5

NT_023148.12

2218266-2218823

14458R

GAT GGC TAT TGA GGA GTA TCC T

14324–14535

5

NT_034772.5

36675175-36674964

33

14339F

ACC CCA TCA TAC TCT TTC ACC C

14550–15042

5

NT_034772.5

1797288-1796796

15052R

TAG GCC TCG CCC GAT GTG TA

14604–15051

5

NT_034772.5

36674895-36674448

34

14971F

TGG CTG AAT CAT CCG CTA CCT T

15041–15785

17

NT_024862.13

336116-336858

15786R

AAA GGG TAG CTT ACT GGT TGT CC

15345–15774

5

NT_023148.12

2219715-2220144

35

15673F

ATC CAA ACA ACA AAG CAT AAT ATT TC

15672–15994

5

NT_023148.12

2220042-2220364

16009R

AAT TAG AAT CTT AGC TTT GGG TG

36

15777F

GCT ACC CCT TCA TCA CCT TC

15785–16388

17

NT_024862.13

336858-337463

16398R

CAA CGG ACC ACT ATC TGA GGG

<p>Figure 4</p>

The distribution of numt clones.

The distribution of numt clones. The distribution of numt clones (based on our primers) across the rCRS reveals regions of the rCRS with multiple numts copies. The pseudo-mitochondrial genome assembled from consensus numt sequences.The distribution of numt clones across the rCRS reveals sites that could be problematic when primers are designed to targets in these regions. Clone name, chromosomal location and rCRS positions are indicated above each clone.

 <p>Figure 5</p>

Our primers recovered lower number of paralogous sequences compared to BLAST searches.

Our primers recovered lower number of paralogous sequences compared to BLAST searches. A BLAST search using the rCRS region covered by the three clones (Figure 3a)returns more numts representative of this region(25)than the three obtained by our cloning data.

Multiple numt copies exist in the genome

To cross-validate our cloned data, we analyzed genomic DNA from ρ0 cells, blood, and human placenta using mitochondrial primers that co-amplified nuclear loci in the prostate cancer specimens. In blood and human placenta samples, a single mtDNA amplicon was consistently observed (Figure 7, and data not shown). Although sequence analysis of the prostate specimens detected numts, their signals were below the detection limit of agarose gel electrophoresis. In contrast, several primers consistently amplified numts from ρ0 cells generating high molecular weight amplifications in addition to the expected mtDNA fragments (Figure 7). These findings confirm the presence of multiple numts loci in the genome and translate into real concern that numts are present in amplifications that produce more that one band or different size amplicons.

<p>Figure 6</p>

A “piggyback” effect resulting from chromosomal copy number and shared divergent sites is demonstrated in a patient sample.

A “piggyback” effect resulting from chromosomal copy number and shared divergent sites is demonstrated in a patient sample. The chromatogram is from a patient for whom heteroplasmy at positions 1709 and 1719 were later noted tobe homologous to three chromosomes (3, 5, and 11), suggesting a possible co-amplification of numts in this instance.

 <p>Figure 7</p>

Multiple numt copies are present in the nucleus

Multiple numt copies are present in the nucleus. PCR amplification of total DNA extracted from ?0 and blood (bld) cells with primers targeting ND1, ATPase6 and CYTB genes. In contrast to the single amplicons obtained from blood, template from ?0 contains additional high molecular weight amplicons. Lad is a 100bp DNA size standard (Fermentas life sciences).

Survey of mitochondrial genome mutations associated with disease suggests caution

Based on our findings that false heteroplasmic sites occurred when there was co-amplification of multiple numt loci with the same nucleotide at that particular site, we compared our cloning data to possible disease associated mutations listed on MITOMAP 52 and common sites were noted. In addition, a BLAST search was performed for these sites and hits held in common between the marker and cloning information were scored as well. Numerous commonalities were noted, which is cause for concern (Table 3).

<p>Table 3</p>

Locus

Disease

Allele

Hom/Het

BLAST hits

Cloning data

Leu

MM/CPEO

T3250C

-/+

C = 16; A = 1

C = 17

Leu

CPEO

C3254T

+/-

T = 18; A = 2

T = 2, 4 and 17

Leu

MELAS

T3291C

-/+

C = 17

C = 2, 4 and 17

ND1

NIDDM; LHON; PEO

G3316A

+/-

A = 17

A = 2, 4, 16, 17

ND1

LHON

G3496T

+/-

T = 14; A = 2

T = X, 2, 4, 16, & 17

ND1

MELAS

G3697A

-/+

A = 3; C = 1

A = 2 & 4; G = X, 16, 17

ND1

Adult-Onset Dystonia

A3796G

-/+

G = 6

A = 2,4 & 17; G = X & 16

ADPD/Hearing Loss &

Gln

Migrane

T4336C

+/+

C = 1

C = 17

ND2

AD;PD

G5460T/A

+/+

T = 5; A = 9

G = 1 (2); T = 2

Asn

CPEO/MM

G5703A

-/+

A = 24

A = 17

COI

PCA

G5913A

+/-

A = 18; T = 1

G = 1(2); A = 17

Myogloinuria; Exercise

CO1

Intolerance

G5920A

-/+

A = 0

G = 1 (2); A = 17

CO1

PCA

G5973A

+/-

A = 3

G = 1(2); A = 17

COI

PCA

G6081A

+/-

A = 1; T = 1

G = 1 (2); A = 17

Ser

MM/Exercise Intolerance

G7497A

+/+

A = 14

A = 5

ATP6

LDYT

G8950A

+/-

A = 2

A = 5

ATP6

Leigh Disease

T9185C

-/+

C = 1

C = 5

ND4

MELAS

A11084G

+/+

G = 9

G = 9

His

MICM

G12192A

+/-

A = 9

G = 5 (3); A = 2(5)

ND5

LHON

G13708A

+/-

A = 15

A = 5 & 9

Paracrystalline Inclusions

CYTB

with Exercise Intolerance

G15497A

+/-

A = 2

A = 17

Thr

Multiple Sclerosis

G15927A

+/-

A = 5

G = 5; A = 17

Type 2 Diabetes;

D-Loop

Cardiomyopathy

T16189C

+/+

C = 0

C = 17

Hom: homoplasmy; Het: heteroplasmy;BLAST hits: number of times a unique numt nucleotide location was returned with a BLAST search; Cloning data: designates chromosome(s) location of nucleotide from cloning data; AD: Alzeimer's Disease; ADPD: Alzeimer's and Parkinson's Disease; PD: Parkinson's Disease; CPEO: Chronic Progressive External Opthalmoplegia; PEO: Progressive External Opthalmoplegia; LDYT: Leber's hereditary optic neuropathy and Dystonia; MELAS: Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes; LHON: Leber's Hereditary Optic Neuropathy; MICM: Maternally Inherited Cardiomyopathy; MM: Mitochondrial Myopathy;NIDDM: Non-Insulin Dependent Diabetes; PCA: Prostate Cancer.

Discussion

In this study, we recovered and assembled the entire mitochondrial genome from nuclear loci. Moreover, this "pseudo-mitochondrial genome" involves numts from over half of the human complement of chromosomes, including the X chromosome. This suggests a widespread allocation of numts in the human nuclear genome. Surprisingly, this distribution was achieved with primers routinely used to amplify mtDNA, yet designed without consideration for numts. Seventy-one percent (24/34) of the primers co-amplified numts in prostate cancer tissue samples. This validates prior suggestions that numts are a potential source of misinformation and serves to illustrate the ease of co-amplification of both mtDNA and nuclear embedded paralogous mitochondrial DNA sequences 16. Our data demonstrate that contrary to a consensus of opinions that the copy number of the mitochondrial genome "swamps" the signal from numts loci, there are circumstances which favor PCR recovery of numts, such as multiple pseudogene copy number 18. For instance, heteroplasmic mutations had been associated with late-onset Alzheimer's disease 1753; however, these false heteroplasmies resulted from co-amplification of numts 195455. Indeed, human numts perplexed ancient DNA studies as well when it was reported that DNA had been recovered and amplified from a Cretaceous dinosaur bone 56. This sequence corresponded to a human numt containing cytochrome b sequence 57, probably from reagent or sample contamination.

Direct pseudogene contribution is not always obvious and can confound suggested mtDNA biomarkers. For example, one set of primers in our data set amplifies tRNAleu and ND1 (3230–3893). Subsequent cloning data identified co-amplification of paralogous numts on five chromosomes with this amplicon. Of specific interest are bases 3316, 3496, 3697 and 3796, which are reported as potential disease associated sites 52. These sites are problematic since the reported base changes are consistent with pseudogene presentation in our data. Further, results from new sequencing technologies have suggested that homoplasmic signals may indeed be heteroplasmic in nature 11. In addition, the heteroplasmic patterns seen at bps 3697 and 3796 are mirrored by the nuclear pseudogene patterns (Table 3).

Re-examination of the raw data from the above studies could address if the disease mutations are actually due to co-amplified numts. Potential markers must be thoroughly investigated to preclude the inclusion of false mutations in the interpretation of mtDNA mutations. BLAST searches of nuclear pseudogenes belie the possibility of widespread integration and/or replication of these sequences, since primers may amplify homologous numts embedded elsewhere in the genome. Thus, high copy numbers for these nuclear segments can produce potential misleading heteroplasmic signals.

Comparative marker studies require the same conditions and primers for meaningful results. For example, proposed MELAS sites T3250C and T3291C (tRNAleu) have paralogous nuclear sites 585960. Follow-up work by Akanuma et al. (2000) 61, demonstrate a corollary numt associated site for T3250C, but not T3291C. A BLAST search shows that these competing sequences are on separate chromosomes (17 and 20) indicating comparison of dissimilar data. In addition, the cloning data here identifies similar regions on chromosomes 2, 4 and 17 (Table 3). Clearly there are numerous paralogous loci for mitochondrial tRNAleu, the amplification of which depends on location and homology of primer sets.

Because of this association in our data, we compared our cloning results to the suggested mitochondrial genome disease associated sites listed on MITOMAP 52. Results suggest that many mutations require meticulous scrutiny because of paralogous nuclear commonalities. Although many of these mutations may well be actual disease markers, the possibility of numt association may confound detection. For example, proposed prostate cancer mutations (G5913A, G5973A and G6081A) are identified, by cloning data, as resident on chromosomes 1 (2 homologous copies) and 17 even though the authors exercised precautionary measures by scanning a database of known nuclear pseudogenes 4962. A locus on chromosome 6 was identified as a potential co-amplification product, yet chromosomes 1 and 17 were not detected. Co-amplification of numts is primer dependent, which may explain the differences seen here; however, database limitations and the absence of extensive wet-lab numt data obscure the meaning of the marker. Particularly suspicious are those sites which demonstrate heteroplasmy in both normal and disease tissue. This may reflect a consistent pattern of numt amplification, an unintended characteristic of primer design. A subset of the marker work prior to the seminal numt work of Lopez et al. (1994) 14 may need clarification as well. For example, an ND2 lesion associated with Alzheimer's and Parkinson's diseases (G5460T/A) has modulated T and A nucleotides. Both mutations are seen in a BLAST search of numts. Moreover, our cloning data also identifies a G at this point, but not a T, again suggesting the relevance of primer selection; nevertheless, all possible modulations are described in this early work (see Kosel 1994. 63), yet it remains a primary reference for this lesion.

If the use of mitochondrial DNA and in particular somatic mitochondrial genome mutations has important utility and medical merit, much of the data requires critical follow-up from a pseudogene perspective. Amplification of ρ0 DNA template with primers to identify and eliminate those which co-amplify nuclear pseudogenes is a vital and necessary procedure 16. For example, mitochondrial PCR protocols were simultaneously run on clinical samples and nucleic acid recovered from ρ0 cells to identify and exclude co-amplification of numts in work by Coskun et al. 64. Alternatively, data may be screened by amplification and sequencing of ρ0 derived DNA and conflicting sites then backed out of actual data generated with identical primers; however, this approach is labor-intensive 43. Phylogenetic analysis of the data would also help distinguish polymorphisms from authentic mutations 22. In general, and unfortunately the advice by Parfait et al. has been largely ignored 16.

Our surprising results are not limited to short amplicons, but are also detected in much larger amplicons. For example, the overlapping amplification of chromosome 5 from bp 8816 to 15051 cautions against assuming that long amplicons are pseudogene free. These possibilities and characteristics of the nuclear genome must be considered when using mitochondrial sequence data for population, forensic or disease studies. Although designing and testing primers to avoid co-amplification of numts is a good laboratory practice, compilation of numts representative of the entire mitochondrial genome is valuable to catalog and characterize the overall nuclear burden of these sequences.

Conclusion

Amplification of overlapping numts paralogous to the mitochondrial genome indicates that co-amplification of nuclear mitochondrial pseudogenes is a real problem for accurate sequence interpretation. Not only is co-amplification dependent on the particular amplicon used, but the copy number of these loci is also important. Only certain positions across the mitochondrial genome are associated with multiple copies of numts. Mitochondrial DNA heteroplasmy should be interpreted with caution since they can be the result of nuclear/cytoplasmic co-amplification. Herein, we have demonstrated the robust amplification of numts. This paper is the first to fully sequence the 46 paralogous DNA fragments that represent the entire mitochondrial genome using 36 primer pairs. This is a surprisingly low number, but reveals that only a limited number of paralogous numts are relevant when considering if heteroplasmic call are authentic mutations. Compilation of a complete data set of numt sequences will help others distinguish paralogous nuclear based heteroplasmy in forensic, population and medical applications.

Methods

Nucleic acid extraction

All research involving human tissue was approved by the Thunder Bay Regional Health Sciences Centre Ethics Committee in accordance with the Tri-Council Policy Statement for Research Involving Humans http://www.nserc.ca/programs/ethics.htm. Archived formalin fixed and paraffin embedded (FFPE) prostatectomy samples were laser capture microdissected (LCM) and DNA isolated by proteinase K digestion. DNA was isolated from blood using the UltraClean™ DNA BloodSpin kit (MO BIO laboratories, Inc). Human placenta DNA was purchased from Sigma-Aldrich (D4642). ρ0cells were prepared from a human osteocarcoma cell line 143B (ATCC CRL-8303) treated with ethidium bromide to deplete cytoplasmic mitochondrial DNA(kindly provided by Eric Shoubridge) 65. Cells were grown to confluence in high glucose DMEM with pyruvate, L-glutamine, uridine (50 μg/ml) and 5% FBS. At confluence cultures were harvested and DNA was extracted using QIAmp DNA Mini Kit.

Amplification, cloning and sequencing

Template from FFPE tissue samples and ρ0 cells were amplified using 34 mitochondrial and 2 chromosome 17 primers. Using TaKaRa LA Taq DNA polymerase (Takara Bio Inc.), PCR reactions were performed using the following conditions: 1X LA PCR Buffer II (Mg2+plus), 0.4 mM each dNTP mixture, 1X BSA (New England Biolabs Inc.), 0.6 μM each primer, 1.25 Units LA Taq, 0.5% Ficoll 400 and 1 mM tartrazine (20,195-2, Aldrich). Total reaction volume was 25 μl. Cycling parameters were 94°C for 2 minutes, followed by 40 cycles of 94°C for 20 seconds, 30 seconds annealing at primer-specific optimized temperatures, and 72°C for 90 seconds. Cycling was performed on a DNA Engine Tetrad 2 (Bio-Rad, Hercules, CA). PCR products were purified, cloned and sequenced at Lark™ Technologies using in-house standard operating procedures (Houston, Texas). In general, 40 clones from each ρ0 amplicon were selected and sequenced in both forward and reverse directions.

Analysis

Sequences were analyzed using the Phred-Phrap-Consed software package 66. The sequences were then grouped based on similarity and a megaBLAST search of NCBI database was performed (using default parameters) to identify all the nuclear co-ordinates of the fragments. This enabled the chromosomal location and nuclear copy number of each amplicon to be determined. Pairwise sequence alignment was performed between the revised Cambridge Reference Sequence (rCRS)67 and the ρ0 clones from the suite of amplicons covering the entire mitochondrial genome using the Sequencher™ software(Gene Codes Corporation).

Southern Blotting

Mitochondrial genomes were cut with PvuII from 2 ug of total DNA extracted from normal blood and ρ0 cells. Digested product was electrophoresed through a 0.4% agarose gel and blotted onto a membrane (Hybond-N+, Roche Applied Sciences). Probes were generated from full length mtDNA (16.5 kb) by random primer labeling using the DIG System (Roche Diagnostics). Blots were incubated with probe, washed, blocked, incubated with anti-digoxigenin-AP fragments (Roche Applied Science) and reacted with a chemiluminescent substrate (CDP-Star®) and exposed to X-ray film (Kodak) as recommended by the DIG Application Manual for Filter Hybridization (Roche Diagnostics, 2000).

PCR

For reverse transcriptase PCR analysis, total RNA was extracted from ρ0 cells and a snap frozen skin sample using standard protocols outlined in the RNeasy Micro Kit manual (Qiagen). A DNase1 treatment step was included in the RNA extraction process to ensure the complete removal of all genomic DNA. We assessed RNA quantity and quality with the ND-1000 spectrophotometer (NanoDrop® technologies) and by gel electrophoresis. First strand DNA was synthesized with the Omniscript® RT (Qiagen) kit. 2 ul of the cDNA was amplified with primer sets to coding mitochondrial genes and a nuclear gene, 5-aminolaevulinate synthase (hALAS) (Table 3), using the PCR conditions described above except the annealing temperature for these primers was 54°C.

To examine for multiple copy numts, 50 ng of genomic DNA from ρ0 cells, blood and human placenta were amplified as described above, using primer sets to OXPHOS genes,.

Authors' contributions

RLP conceived of and supervised the study, and drafted the manuscript, JM, BR AA, and KR conducted experiments and participated in sequence alignment and data analysis, RW co-coordinated sample collection, GDD conducted experiments and helped draft the manuscript, JPJ provided intellectual insight and helped draft the manuscript, RET designed experiment and helped draft manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank Eric Shoubridge for ρ0 cells and Lark Technologies for cloning and sequencing. Financial support for this study was provided to Genesis Genomics Inc. by Industry Canada (FedNor), National Research Council-Industrial Research Assistance Program (NRC-IRAP), and Northern Ontario Heritage Fund Corporation (NOHFC).

 <p>The DNA revolution in population genetics</p> Cavalli-Sforza LL Trends Genet 1998 14 2 60 65 10.1016/S0168-9525(97)01327-9 9520599 <p>Mitochondrial DNA and Human Evolution</p> Pakendorf B Stoneking M Annu Rev Genomics Hum Genet 2005 16124858 <p>Forensics and mitochondrial DNA: applications, debates, and foundations</p> Budowle B Allard MW Wilson MR Chakraborty R Annu Rev Genomics Hum Genet 2003 4 119 141 10.1146/annurev.genom.4.070802.110352 14527299 <p>Mitochondrial DNA as a potential tool for early cancer detection</p> Parr RL Dakubo GD Thayer RE McKenney K Birch-Machin MA Hum Genomics 2006 2 4 252 257 16460650 <p>Facile detection of mitochondrial DNA mutations in tumors and bodily fluids</p> Fliss MS Usadel H Caballero OL Wu L Buta MR Eleff SM Jen J Sidransky D Science 2000 287 5460 2017 2019 10.1126/science.287.5460.2017 10720328 <p>On respiratory impairment in cancer cells</p> Warburg O Science 1956 124 3215 269 270 13351639 <p>Mitochondrial defects in cancer</p> Carew JS Huang P Mol Cancer 2002 1 1 9 149412 12513701 10.1186/1476-4598-1-9 <p>Mitochondrial DNA alterations in cancer</p> Copeland WC Wachsman JT Johnson FM Penta JS Cancer Invest 2002 20 4 557 569 10.1081/CNV-120002155 12094550 <p>Mitochondrial DNA in human malignancy</p> Penta JS Johnson FM Wachsman JT Copeland WC Mutat Res 2001 488 2 119 133 10.1016/S1383-5742(01)00053-9 11344040 <p>Mitochondrial DNA mutations in human disease</p> Taylor RW Turnbull DM Nat Rev Genet 2005 6 5 389 402 10.1038/nrg1606 15861210 <p>Mitochondrial DNA as a cancer biomarker</p> Jakupciak JP Wang W Markowitz ME Ally D Coble M Srivastava S Maitra A Barker PE Sidransky D O'Connell CD J Mol Diagn 2005 7 2 258 267 15858150 <p>Standards for validation of cancer biomarkers</p> O'Connell CD Atha DH Jakupciak JP Cancer Biomarkers 2005 1 233 239 <p>Mitochondrial DNA mutations in human neoplasia</p> Czarnecka AM Golik P Bartnik E J Appl Genet 2006 47 1 67 78 16424612 <p>Numt, a recent transfer and tandem amplification of mitochondrial DNA to the nuclear genome of the domestic cat</p> Lopez JV Yuhki N Masuda R Modi W O'Brien SJ J Mol Evol 1994 39 2 174 190 7932781 <p>Rates of DNA duplication and mitochondrial DNA insertion in the human genome</p> Bensasson D Feldman MW Petrov DA J Mol Evol 2003 57 3 343 354 10.1007/s00239-003-2485-7 14629044 <p>Co-amplification of nuclear pseudogenes and assessment of heteroplasmy of mitochondrial DNA mutations</p> Parfait B Rustin P Munnich A Rotig A Biochem Biophys Res Commun 1998 247 1 57 59 10.1006/bbrc.1998.8666 9636653 <p>Mutations in mitochondrial cytochrome c oxidase genes segregate with late-onset Alzheimer disease</p> Davis RE Miller S Herrnstadt C Ghosh SS Fahy E Shinobu LA Galasko D Thal LJ Beal MF Howell N Proc Natl Acad Sci USA 1997 94 9 4526 4531 20756 9114023 10.1073/pnas.94.9.4526 <p>Apparent mtDNA heteroplasmy in Alzheimer's disease patients and in normals due to PCR amplification of nucleus-embedded mtDNA pseudogenes</p> Hirano M Shtilbans A Mayeux R Davidson MM DiMauro S Knowles JA Schon EA Proc Natl Acad Sci USA 1997 94 26 14894 14899 25134 9405710 10.1073/pnas.94.26.14894 <p>Evidence that two reports of mtDNA cytochrome c oxidase "mutations" in Alzheimer's disease are based on nDNA pseudogenes of recent evolutionary origin</p> Davis JN 2nd Parker WD Jr Biochem Biophys Res Commun 1998 244 3 877 883 10.1006/bbrc.1998.8353 9535760 <p>Problems in FBI mtDNA database</p> Bandelt HJ Salas A Bravi C Science 2004 305 5689 1402 1404 10.1126/science.305.5689.1402b 15353782 <p>A call for mtDNA data quality control in forensic science</p> Yao YG Bravi CM Bandelt HJ Forensic Sci Int 2004 141 1 1 6 10.1016/j.forsciint.2003.12.004 15066707 <p>A critical reassessment of the role of mitochondria in tumorigenesis</p> Salas A Yao YG Macaulay V Vega A Carracedo A Bandelt HJ PLoS Med 2005 2 11 e296 1240051 16187796 10.1371/journal.pmed.0020296 <p>A practical guide to mitochondrial DNA error prevention in clinical, forensic, and population genetics</p> Salas A Carracedo A Macaulay V Richards M Bandelt HJ Biochem Biophys Res Commun 2005 335 3 891 899 10.1016/j.bbrc.2005.07.161 16102729 <p>Phantom mutation hotspots in human mitochondrial DNA</p> Brandstatter A Sanger T Lutz-Bonengel S Parson W Beraud-Colomb E Wen B Kong QP Bravi CM Bandelt HJ Electrophoresis 2005 26 18 3414 3429 10.1002/elps.200500307 16167362 <p>A reappraisal of complete mtDNA variation in East Asian families with hearing impairment</p> Yao YG Salas A Bravi CM Bandelt HJ Hum Genet 2006 16528519 <p>The Human MitoChip: a high-throughput sequencing microarray for mitochondrial mutation detection</p> Maitra A Cohen Y Gillespie SE Mambo E Fukushima N Hoque MO Shah N Goggins M Califano J Sidransky D Genome Res 2004 14 5 812 819 479107 15123581 10.1101/gr.2228504 <p>Mitochondrial diabetes: investigation and identification of a novel mutation</p> Lynn S Wardell T Johnson MA Chinnery PF Daly ME Walker M Turnbull DM Diabetes 1998 47 11 1800 1802 9792552 <p>Mitochondrial DNA and disease</p> Dimauro S Davidzon G Ann Med 2005 37 3 222 232 10.1080/07853890510007368 16019721 <p>Accurate detection and quantitation of heteroplasmic mitochondrial point mutations by pyrosequencing</p> White HE Durston VJ Seller A Fratter C Harvey JF Cross NC Genet Test 2005 9 3 190 199 10.1089/gte.2005.9.190 16225398 <p>Mitochondrial DNA analysis in clinical laboratory diagnostics</p> Wong LJ Boles RG Clin Chim Acta 2005 354 1–2 1 20 10.1016/j.cccn.2004.11.003 15748595 <p>Extremely high levels of human mitochondrial DNA heteroplasmy in single hair roots</p> Grzybowski T Electrophoresis 2000 21 3 548 553 10.1002/(SICI)1522-2683(20000201)21:3<548::AID-ELPS548>3.0.CO;2-U 10726758 <p>Mitochondrial DNA depletion analysis by pseudogene ratioing</p> Swerdlow RH Redpath GT Binder DR Davis JN 2nd VandenBerg SR J Neurosci Methods 2006 150 2 265 271 10.1016/j.jneumeth.2005.06.023 16118020 <p>Mitochondrial DNA mutations and mitochondrial DNA depletion in gastric cancer</p> Wu CW Yin PH Hung WY Li AF Li SH Chi CW Wei YH Lee HC Genes Chromosomes Cancer 2005 44 1 19 28 10.1002/gcc.20213 15892105 <p>Mitochondrial DNA mutations and mitochondrial DNA depletion in breast cancer</p> Tseng LM Yin PH Chi CW Hsu CY Wu CW Lee LM Wei YH Lee HC Genes Chromosomes Cancer 2006 <p>Mitochondrial DNA determines androgen dependence in prostate cancer cell lines</p> Higuchi M Kudo T Suzuki S Evans TT Sasaki R Wada Y Shirakawa T Sawyer JR Gotoh A Oncogene 2006 25 10 1437 1445 10.1038/sj.onc.1209190 16278679 <p>Mitochondrial Genome Instability and mtDNA Depletion in Human Cancers</p> Lee HC Yin PH Lin JC Wu CC Chen CY Wu CW Chi CW Tam TN Wei YH Ann N Y Acad Sci 2005 1042 109 122 10.1196/annals.1338.011 15965052 <p>Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell</p> Satoh M Kuroiwa T Exp Cell Res 1991 196 1 137 140 10.1016/0014-4827(91)90467-9 1715276 <p>Analysis of mtDNA copy number and composition of single mitochondrial particles using flow cytometry and PCR</p> Cavelier L Johannisson A Gyllensten U Exp Cell Res 2000 259 1 79 85 10.1006/excr.2000.4949 10942580 <p>Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells</p> Robin ED Wong R J Cell Physiol 1988 136 3 507 513 10.1002/jcp.1041360316 3170646 <p>Nuclear insertion sequences of mitochondrial DNA predominate in hair but not in blood of elephants</p> Greenwood AD Paabo S Mol Ecol 1999 8 1 133 137 10.1046/j.1365-294X.1999.00507.x 9919702 <p>Unreliable mtDNA data due to nuclear insertions: a cautionary tale from analysis of humans and other great apes</p> Thalmann O Hebler J Poinar HN Paabo S Vigilant L Mol Ecol 2004 13 2 321 335 10.1046/j.1365-294X.2003.02070.x 14717890 <p>The problems and promise of DNA barcodes for species diagnosis of primate biomaterials</p> Lorenz JG Jackson WE Beck JC Hanner R Philos Trans R Soc Lond B Biol Sci 2005 360 1462 1869 1877 10.1098/rstb.2005.1718 16214744 <p>Somatic Mitochondrial DNA Mutations in Prostate Cancer and Normal Appearing Adjacent Glands in Comparison to Age-Matched Prostate Samples without Malignant Histology</p> Parr RL Dakubo GD Crandall KA Maki J Reguly B Aguirre A Wittock R Robinson K Alexander JS Birch-Machin MA J Mol Diagn 2006 8 3 312 319 16825503 <p>A high frequency of sequence alterations is due to formalin fixation of archival specimens</p> Williams C Ponten F Moberg C Soderkvist P Uhlen M Ponten J Sitbon G Lundeberg J Am J Pathol 1999 155 5 1467 1471 10550302 <p>Effect of fixatives and tissue processing on the content and integrity of nucleic acids</p> Srinivasan M Sedmak D Jewell S Am J Pathol 2002 161 6 1961 1971 12466110 <p>Structure and chromosomal distribution of human mitochondrial pseudogenes</p> Tourmen Y Baris O Dessen P Jacques C Malthiery Y Reynier P Genomics 2002 80 1 71 77 10.1006/geno.2002.6798 12079285 <p>Continued colonization of the human genome by mitochondrial DNA</p> Ricchetti M Tekaia F Dujon B PLoS Biol 2004 2 9 E273 515365 15361937 10.1371/journal.pbio.0020273 <p>NUMTs in sequenced eukaryotic genomes</p> Richly E Leister D Mol Biol Evol 2004 21 6 1081 1084 10.1093/molbev/msh110 15014143 <p>Mitochondrial DNA-like sequences in the nucleus (NUMTs): insights into our African origins and the mechanism of foreign DNA integration</p> Mishmar D Ruiz-Pesini E Brandon M Wallace DC Hum Mutat 2004 23 2 125 133 10.1002/humu.10304 14722916 <p>Pattern of organization of human mitochondrial pseudogenes in the nuclear genome</p> Woischnik M Moraes CT Genome Res 2002 12 6 885 893 1383742 12045142 10.1101/gr.227202. Article published online before print in May 2002 <p>A quick, direct method that can differentiate expressed mitochondrial genes from their nuclear pseudogenes</p> Collura RV Auerbach MR Stewart CB Curr Biol 1996 6 10 1337 1339 10.1016/S0960-9822(02)70720-3 8939570 <p>MITOMAP: a human mitochondrial genome database – 2004 update</p> Brandon MC Lott MT Nguyen KC Spolim S Navathe SB Baldi P Wallace DC Nucleic Acids Res 2005 33 Database D611 613 540033 15608272 10.1093/nar/gki079 <p>Multiplex fluorescence-based primer extension method for quantitative mutation analysis of mitochondrial DNA and its diagnostic application for Alzheimer's disease</p> Fahy E Nazarbaghi R Zomorrodi M Herrnstadt C Parker WD Davis RE Ghosh SS Nucleic Acids Res 1997 25 15 3102 3109 146869 9224611 10.1093/nar/25.15.3102 <p>Ancient mtDNA sequences in the human nuclear genome: a potential source of errors in identifying pathogenic mutations</p> Wallace DC Stugard C Murdock D Schurr T Brown MD Proc Natl Acad Sci USA 1997 94 26 14900 14905 25135 9405711 10.1073/pnas.94.26.14900 <p>A novel mitochondrial DNA-like sequence in the human nuclear genome</p> Herrnstadt C Clevenger W Ghosh SS Anderson C Fahy E Miller S Howell N Davis RE Genomics 1999 60 1 67 77 10.1006/geno.1999.5907 10458912 <p>DNA sequence from Cretaceous period bone fragments</p> Woodward SR Weyand NJ Bunnell M Science 1994 266 5188 1229 1232 7973705 <p>Insertions and duplications of mtDNA in the nuclear genomes of Old World monkeys and hominoids</p> Collura RV Stewart CB Nature 1995 378 6556 485 489 10.1038/378485a0 7477403 <p>Diagnosis of mitochondrial disease: assessment of mitochondrial DNA heteroplasmy in blood</p> Taylor RW Taylor GA Morris CM Edwardson JM Turnbull DM Biochem Biophys Res Commun 1998 251 3 883 887 10.1006/bbrc.1998.9553 9791004 <p>A novel point mutation in the mitochondrial tRNA(Leu)(UUR) gene in a family with mitochondrial myopathy</p> Goto Y Tojo M Tohyama J Horai S Nonaka I Ann Neurol 1992 31 6 672 675 10.1002/ana.410310617 1514779 <p>A new point mutation at nucleotide pair 3291 of the mitochondrial tRNA(Leu(UUR)) gene in a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS)</p> Goto Y Tsugane K Tanabe Y Nonaka I Horai S Biochem Biophys Res Commun 1994 202 3 1624 1630 10.1006/bbrc.1994.2119 7520241 <p>Two pathogenic point mutations exist in the authentic mitochondrial genome, not in the nuclear pseudogene</p> Akanuma J Muraki K Komaki H Nonaka I Goto Y J Hum Genet 2000 45 6 337 341 10.1007/s100380070004 11185741 <p>mtDNA mutations increase tumorigenicity in prostate cancer</p> Petros JA Baumann AK Ruiz-Pesini E Amin MB Sun CQ Hall J Lim S Issa MM Flanders WD Hosseini SH Proc Natl Acad Sci USA 2005 102 3 719 724 545582 15647368 10.1073/pnas.0408894102 <p>No association of mutations at nucleotide 5460 of mitochondrial NADH dehydrogenase with Alzheimer's disease</p> Kosel S Egensperger R Mehraein P Graeber MB Biochem Biophys Res Commun 1994 203 2 745 749 10.1006/bbrc.1994.2245 8093052 <p>Alzheimer's brains harbor somatic mtDNA control-region mutations that suppress mitochondrial transcription and replication</p> Coskun PE Beal MF Wallace DC Proc Natl Acad Sci USA 2004 101 29 10726 10731 490002 15247418 10.1073/pnas.0403649101 <p>Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation</p> King MP Attardi G Science 1989 246 4929 500 503 2814477 <p>PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing</p> Nickerson DA Tobe VO Taylor SL Nucleic Acids Res 1997 25 14 2745 2751 146817 9207020 10.1093/nar/25.14.2745 <p>Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA</p> Andrews RM Kubacka I Chinnery PF Lightowlers RN Turnbull DM Howell N Nat Genet 1999 23 2 147 10.1038/13779 10508508