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Differences in tissue distribution of HV2 length heteroplasmy in mitochondrial DNA between mothers and children
Forensic Science International 175 (2008) 155–159 www.elsevier.com/locate/forsciint
Differences in tissue distribution of HV2 length heteroplasmy in mitochondrial DNA between mothers and children Masaru Asari *, Jun-ichi Azumi, Keiko Shimizu, Hiroshi Shiono Department of Legal Medicine, Asahikawa Medical College, 2-1 Midorigaokahigashi, Asahikawa 078-8510, Japan Received 9 November 2006; received in revised form 15 January 2007; accepted 9 June 2007 Available online 23 July 2007
Abstract Sequence analysis of HV2 in mitochondrial DNA has been performed as a tool for forensic identification, in addition to that of HV1. HV2 contains length heteroplasmy, which shows high variability within an individual or in maternal relatives. In this study, we used cloning analysis and PCR direct sequencing to compare, between mothers and their children, HV2 length heteroplasmic profiles in different tissues. For two mother– child pairs, different types of variant distribution were observed by cloning analysis. In pair 1, length heteroplasmic patterns in most tissues were similar (predominantly 9 and 10Cs variants), but different length heteroplasmic levels, with shifts in predominant genotype, were observed for some hairs in both mother and child. In pair 2, genotype distribution was similar for all tissues, with a predominant 8Cs genotype, but varying in the proportion of minor component. The proportion of one minor length variant (9Cs) in blood from the child was significantly higher than that from the mother, but the proportions of minor components (7 and/or 9Cs) in other tissue samples decreased from mother to child. Moreover, we could confirm that sequence types of PCR products were reflected by the distribution of length variants, which were observed especially in high proportion, in cloning analysis. Our results reveal variable changes in length heteroplasmic level in various tissues between generations. Variability between tissues, especially among hairs, within an individual would result in complicated differences in genotype distribution between maternal generations, and correlate with longer length of Cs for predominant variants. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Mitochondrial DNA; Hypervariable region 2; Length heteroplasmy; Tissue distribution
1. Introduction Sequence analysis of hypervariable regions (HV1, 2 and 3) of mitochondrial DNA (mtDNA), which takes advantage of high copy number per cell, maternal inheritance, lack of recombination and high mutation rate, has been routinely performed in forensic identification for a number of years. In these regions, some kinds of length heteroplasmies, which occur by replication slippage and consist of homopolymeric C-tract or CA repeats, are located at the positions 16180–16193 for HV1 [1], 303–315 for HV2 [2], 514–523 and 568–573 for HV3 [3,4]. HV1 and HV2 length heteroplasmic profiles have been analyzed in detail. The HV1 length heteroplasmy contains a homopolymeric cytosine tract caused by a thymine to cytosine transition at position 16189. However, while this HV1 length heteroplasmy
* Corresponding author. Tel.: +81 166 68 2433; fax: +81 166 68 2439. E-mail address: [email protected] (M. Asari). 0379-0738/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2007.06.015
has been observed, it remains difficult to determine sequence type (e.g., the number of cytosines) in this area by PCR direct sequencing, with the exception of the number of adenines preceding the polycytosine [5]. The HV2 length heteroplasmy is at nucleotide positions 303–315 and consists of seven cytosines followed by a thymine followed by five more cytosines, but five cytosines in the second C-stretch are rarely identified, with six cytosines in this region usually being observed. The HV2 length heteroplasmy is classified into two different types, the first is occurred by cytosine insertion/deletion(s) at the positions 303– 309, and the second caused by a thymine to cytosine transition at position 310 [1,6,7]. Although the former type is frequently observed, the latter is quite rare, thus far observed in only 3 out of 120 Europeans [7]. In cases where the proportion of minor cytosine length variants is much lower than that of a major variant, a predominant HV2 sequence type at 303–315 can be identified by direct sequencing analysis [7]. HV1 length heteroplasmic profiles show wide variation in unrelated individuals [8], but seem to be maintained within
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each lineage [9,10] and in various tissues within individuals [7,11]. On the other hand, instability of the HV2 length heteroplasmy has been reported between one or a few generations [12–14]. In particular, stability dramatically decreases when a predominant genotype contains more than eight cytosines in the first C-stretch [9]. Furthermore, different genotype patterns have been observed between and within tissues [15,16]. Consequently, HV2 length heteroplasmy can be intensively complicated and may be difficult to interpret in different tissues of maternal lineages. In recent years, fragment analyses based on PCR using fluorescently-labeled primers have been introduced as highaccuracy forensic techniques [7,16,17]. Although cloning analysis is a cost and time-consuming method and may not be suitable for application in routine forensic cases, it can reveal the components of length variants and their proportions in detail. Consequently, we used cloning analysis and PCR direct sequencing here to examine HV2 length heteroplasmic profiles between mothers and their children, in order to investigate intra- and intergenerational profile differences.
and 2.5 U Pfu DNA polymerase in 1 PCR buffer (Stratagene, Gebouw, CA). Amplification was conducted in a TaKaRa PCR Thermal Cycler Personal (TaKaRa, Otsu, Japan) using the following conditions: (1) denaturation at 95 8C for 2 min, (2) amplification for 32 cycles at 94 8C for 1 min, 55 8C for 1 min, and 72 8C for 1 min, and (3) final extension at 72 8C for 7 min. PCR products were purified by MinElute PCR Amplification Kit (QIAGEN) to remove unconsumed primers and dNTPs.
2. Materials and methods
3. Results and discussion
2.1. Samples and DNA extraction
In this study, we observed different tissue distributions of HV2 length heteroplasmy between and within two mother– child pairs. To minimize possible PCR errors, each reaction was performed using proof-reading DNA polymerase instead of Taq DNA polymerase [7]. In the cloning analysis, 20 clones obtained by two independently generated PCR products were compared and the results obtained from the different products for each sample showed a quite similar distribution. Length variant distribution based on cloning analysis and sequence types of PCR products for pair 1 are shown in Table 1. By cloning analysis, observed variant types ranged from 7 to 13Cs and 15Cs, and profiles in most tissues, except for some hairs, were apparently heteroplasmic with 9 and 10Cs in high proportion. On the other hand, a slightly heteroplasmic profile, consisting of 9Cs as a major variant and minor length components in varying proportion, was observed in one hair
Peripheral blood, buccal cells, nails and four telogen hairs were collected from each member of two mother–child pairs, all of whom exhibited HV2 length heteroplasmy in blood samples and consisted of a variable number of C residues before T at the position 310. Each hair sample was cut to approximately 1 cm in length including the hair root. DNA extractions from blood and buccal cells were performed by QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany), and from nails and hairs by ISOHAIR (NIPPONGENE, Tokyo, Japan), according to the manufacturer’s instructions. The concentrations of DNA from bloods and buccal cells were determined by Nano Drop ND-1000 (Nano Drop Technologies, Wilmington, DE).
2.2. Amplification Amplification of HV2 was performed using primers L29-H408 [18]. The HV2 segment was amplified in a 25 mL total reaction volume containing 5– 20 ng genomic DNA from bloods and buccal cells or a 5-aliquot of the DNA suspension from nails and hair samples, 0.2 mM of each primer, 200 mM dNTP,
2.3. Cloning PCR products were cloned into pGEM-T Easy Vector (Promega, Madison, WI). Plasmid DNA isolation was performed using QIAprep Spin Miniprep Kit (QIAGEN). For each tissue sample, two independently generated PCR products were cloned and a total of 40 clones (20 clones per PCR product) were used.
2.4. Sequencing Plasmid DNA or purified PCR product was directly sequenced using BigDye Terminator Cycle Sequencing Kit v1.1 (Applied Biosystems, Foster City, CA) according to the manufacturer’s manual. All sequencing reactions were analyzed using the ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Sequences were aligned to the revised Cambridge reference sequence [19].
Table 1 Distribution of length variants and sequence types downstream of position 303 for pair 1 Mother
Variants of Cs 7 8 9 10 11 12 13 15 Sequence type of PCR producta a
Child
Blood
Buccal cells
Nails
Hair1
Hair2
Hair3
Hair4
Blood
Buccal cells
Nails
Hair1
Hair2
Hair3
Hair4
0 1 18 16 4 1 0 0 Ia
0 0 14 22 4 0 0 0 Ia
1 5 13 15 5 1 0 0 Ia
0 3 14 15 3 5 0 0 Ia
1 0 12 15 4 5 1 2 Ia
0 1 19 16 4 0 0 0 Ia
2 5 20 7 3 2 1 0 Ib
0 1 20 18 0 1 0 0 Ia
2 3 15 17 3 0 0 0 Ia
0 2 12 17 9 0 0 0 Ia
0 1 36 3 0 0 0 0 Ic
0 3 33 4 0 0 0 0 Ic
0 0 22 17 1 0 0 0 Ia
0 5 19 13 2 1 0 0 Ia
Categorized into three characteristic types (Type Ia, Ib and Ic). Type Ia does not contain a predominant genotype and shows severely blurred sequences downstream of the first C-stretch within 303–315. Type Ib shows a predominant 9Cs genotype and the sequence downstream of the second C-stretch within 303–315 is unreadable. Type Ic contains a predominant 9Cs genotype and no unreadable sequence.
M. Asari et al. / Forensic Science International 175 (2008) 155–159
Fig. 1. Sequence electropherograms between positions 300 and 320 of representative examples for the three types observed in pair 1. Arrows indicate sites of both T and C peaks observed in equally high proportion.
sample (Hair4) from mother and two hairs (Hair1, 2) from child. Especially in the two hairs from child, very low levels of both the 8 and 10Cs variants were observed (1–4 clones). Corresponding to the distribution of length variants observed in each sample, three characteristic patterns (Type Ia, Ib and Ic) downstream of position 303 were identified by direct sequencing analysis of PCR products. Electropherograms of representative examples for the three types are shown in Fig. 1. In most samples except for Hair4 from the mother and Hairs 1 and 2 from the child, a predominant genotype cannot be identified; what we observed nine unambiguous C peaks followed by both T and C peaks in equally high proportion at the next two consecutive positions
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(Type Ia). The remaining three hair samples commonly showed a predominant 9Cs genotype, with the sequence downstream of the second C-stretch within 303–315 being unreadable in Hair4 from the mother (Type Ib), but identifiable in Hairs 1 and 2 from the child (Type Ic). Although the heteroplasmic patterns for most tissues in pair 1 were quite similar, the strikingly different length heteroplasmic levels observed for some hairs suggest the existence of significant variation among hairs and between tissues within an individual. In general, differences in length heteroplasmic profiles has been explained as a result of a genetic bottleneck due to segregation of the mtDNA, and a larger bottleneck would result in fixed or similar profiles [6,12,16]. For hairs in particular, formation of each hair root and shaft derives from discrete groups of stem cells, compared to other samples such as blood or organ tissues [16,20,21]. Therefore, variable length heteroplasmic levels, with shifts in predominant genotype, could be observed among hairs or across other tissues within an individual, resulting in variability between maternal generations. Furthermore, several studies have shown that differences of point or length heteroplasmy were confirmed not only between hairs and the references (e.g., blood or buccal cells), but also among independent extracts from one hair, both within and between laboratories [22–24]. In addition to the effect of methodology including detection reagents, it has been demonstrated that differential segregation of heteroplasmic variants are observed in various sections of a single hair, and the existence of these differences should be considered in analysis of hair samples. The results for pair 2 are shown in Table 2. By cloning analysis, variant types consisting of 7–11 consecutive Cs were identified, and profiles of all tissues in both mother and child consisted of predominantly 8Cs variants, but varying in the proportion of minor components. As minor components, 7 and 9Cs were identified in blood and hair samples. On the other hand, 9 and 10Cs were commonly observed in buccal cells and nails, and nails from the mother and buccal cells from the child contained 11 and 7Cs, respectively. Furthermore, the profile in one hair (Hair4) from the child is purely homoplasmic, consisting of 8Cs variants alone. With respect to tissue distribution within an
Table 2 Distribution of length variants and sequence types downstream of position 303 for pair 2 Mother
Variants of Cs 7 8 9 10 11 Sequence type of PCR producta
Child
Blood
Buccal cells
Nails
Hair1
Hair2
Hair3
Hair4
Blood
Buccal cells
Nails
Hair1
Hair2
Hair3
Hair4
1 38 1 0 0 IIa
0 32 7 1 0 IIa
0 21 13 5 1 IIc
7 32 1 0 0 IIb
2 36 2 0 0 IIa
8 29 3 0 0 IIb
3 32 5 0 0 IIa
1 31 8 0 0 IIa
1 34 4 1 0 IIa
0 26 12 2 0 IIc
1 39 0 0 0 IIa
0 39 1 0 0 IIa
0 37 3 0 0 IIa
0 40 0 0 0 IIa
a Categorized into three characteristic types (Type IIa, IIb and IIc). Type IIa shows a predominant 8Cs genotype and contains no unreadable sequence in HV2. Type IIb and IIc do not show a predominant genotype, and contain blurred sequence downstream of the first C-stretch within 303–315 and different patterns of multiple T peaks, which interrupt the polycytosine within 303–315. Type IIb contains the pattern with the highest T peak following the second highest T peak, but type IIc contains the pattern with the highest T peak preceding the second highest.
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individual, the proportions of minor components in nails were significantly higher than in other tissues. Sequences downstream of position 303 for pair 2 by direct sequencing were also classified into three types (Type IIa, IIb and IIc). In most samples, except for nails from both mother and child, and two hairs (Hair1, 3) from mother, C8 genotypes and full-length HV2 sequences were determined (Type IIa). Severely blurred sequence downstream of the first C-stretch was observed in the remaining four samples, and thus a predominant C genotype could not be determined. Moreover, these samples were subdivided into two types based on patterns of multiple T peaks that interrupt the polycytosine within positions 303–315; type IIb contains the pattern with the highest T peak following the second highest T peak, and was observed in nails from both mother and child, but type IIc, which was observed in two hairs (Hair1, 3) from mother, contains the pattern with the highest T peak preceding the second highest. Electropherograms of representative examples for these types are shown in Fig. 2. As in the case of pair 1, we could confirm that the electropherogram for each sample of pair 2 was reflected by the distribution of length variants. As shown by the results for pair 2, a variety of profiles are not necessarily observed among hairs within an individual; profile variety may be associated with longer length of Cs of a predominant genotype in samples such as blood or other organ tissues. However, length-of-Cs genotypes may not strictly correlate with stable or unstable genotype distribution between tissues, including among hairs. In one study, shifts in the
genotype patterns have been observed among hairs in an individual with a 8Cs genotype in blood samples [16]. In terms of maternal distribution in pair 2, the proportion of a minor length variant (9Cs) in blood from the child was significantly higher than that from the mother and the proportions of 7 and/or 9Cs were smaller in the child compared with those of the mother in other tissue samples. These results indicate that different intergenerational shifts of heteroplasmic level across tissues can occur. More complicated qualitative distribution also should be identifiable in various tissues of maternally related members, given that rapid shifts in predominant genotype have been observed between one or a few generations [6,13]. In the forensic field, the existence of heteroplasmy, and differences in the proportion of components between generations or within individuals, has made interpretation of mtDNA sequences more difficult. As shown in the published guidelines [25] and recommendations [26], determination of the number of cytosines is difficult and the exact number should not be considered, if a characteristic ‘‘out-of-phase’’ pattern downstream of the first C-stretch within positions 303–315 is observed. However, profiles lacking the exact number of cytosines sometimes contain enough information to be considered useful for identification purposes. In terms of leading to a decision (inclusive, exclusive or inconclusive), comparison of length heteroplasmic profiles can be valuable and informative. In conclusion, our results reveal variable changes in length heteroplasmic level in various tissues between generations. Variability between tissues, especially among hairs, within an individual would result in complicated differences in genotype distribution between maternal generations, and correlate with longer length of Cs for predominant variants. References
Fig. 2. Sequence electropherograms between positions 300 and 320 of representative examples for the three types observed in pair 2. Arrows indicate sites of both T and C peaks observed in equally high proportion.
[1] K.E. Bendall, B.C. Sykes, Length heteroplasmy in the first hypervariable segment of the human mtDNA control region, Am. J. Hum. Genet. 57 (1995) 248–256. [2] B.D. Greenberg, J.E. Newbold, A. Sugino, Intraspecific nucleotide sequence variability surrounding the origin of replication in human mitochondrial DNA, Gene 21 (1983) 33–49. [3] A. Bodenteich, L.G. Mitchell, M.H. Polymeropoulos, C.R. Merril, Dinucleotide repeat in the human mitochondrial D-loop, Hum. Mol. Genet. 1 (1992) 140. [4] A. Torroni, M.T. Lott, M.F. Cabell, Y.S. Chen, L. Lavergne, D.C. Wallace, mtDNA and the origin of Caucasians: identification of ancient Caucasianspecific haplogroups, one of which is prone to a recurrent somatic duplication in the D-loop region, Am. J. Hum. Genet. 55 (1994) 760–776. [5] C. Bini, G. Pappalardo, mtDNA HVI length heteroplasmic profile in different tissues of maternally related members, Forensic Sci. Int. 152 (2005) 35–38. [6] J.E. Stewart, C.L. Fisher, P.J. Aagaard, M.R. Wilson, A.R. Isenberg, D. Polanskey, E. Pokorak, J.A. DiZinno, B. Budowle, Length variation in HV2 of the human mitochondrial DNA control region, J. Forensic Sci. 46 (2001) 862–870. [7] S. Lutz-Bonengel, T. Sanger, S. Pollak, R. Szibor, Different methods to determine length heteroplasmy within the mitochondrial control region, Int. J. Legal Med. 118 (2004) 274–281. [8] H.Y. Lee, U. Chung, J.E. Yoo, M.J. Park, K.J. Shin, Quantitative and qualitative profiling of mitochondrial DNA length heteroplasmy, Electrophoresis 25 (2004) 28–34.
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[19] R.M. Andrews, I. Kubacka, P.F. Chinnery, R.N. Lightowlers, D.M. Turnbull, N. Howell, Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA, Nat. Genet. 23 (1999) 47. [20] C.A. Linch, D.A. Whiting, M.M. Holland, Human hair histogenesis for the mitochondrial DNA forensic scientist, J. Forensic Sci. 46 (2001) 844–853. [21] K.E. Bendall, V.A. Macaulay, B.C. Sykes, Variable levels of a heteroplasmic point mutation in individual hair roots, Am. J. Hum. Genet. 61 (1997) 1303–1308. [22] G. Tully, S.M. Barritt, K. Bender, E. Brignon, C. Capelli, N. DimoSimonin, C. Eichmann, C.M. Ernst, C. Lambert, M.V. Lareu, B. Ludes, B. Mevag, W. Parson, H. Pfeiffer, A. Salas, P.M. Schneider, E. Staalstrom, Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts, Forensic Sci. Int. 140 (2004) 1–11. [23] A. Alonso, A. Salas, C. Albarran, E. Arroyo, A. Castro, M. Crespillo, A.M. Di Lonardo, M.V. Lareu, C.L. Cubrija, M.L. Soto, J.A. Lorente, M.M. Semper, A. Palacio, M. Paredes, L. Pereira, A.P. Lezaun, J.P. Brito, A. Sala, M.C. Vide, M. Whittle, J.J. Yunis, J. Go´mez, Results of the 1999– 2000 Collaborative Exercise and proficiency testing program of mitochondrial DNA of the GEP–ISFG: an inter-laboratory study of the observed variability in the heteroplasmy level of hair from the same donor, Forensic Sci. Int. 125 (2002) 1–7. [24] A. Salas, M.V. Lareu, A. Carracedo, Heteroplasmy in mtDNA and the weight of evidence in forensic mtDNA analysis: a case report, Int. J. Legal Med. 114 (2001) 186–190. [25] G. Tully, W. Ba¨r, B. Brinkmann, A. Carracedo, P. Gill, N. Morling, W. Parson, P. Schneider, Considerations by the European DNA profiling (EDNAP) group on the working practices, nomenclature and interpretation of mitochondrial DNA profiles, Forensic Sci. Int. 124 (2001) 83–91. [26] A. Carracedo, W. Ba¨r, P. Lincoln, W. Mayr, N. Morling, B. Olaisen, P. Schneider, B. Budowle, B. Brinkmann, P. Gill, M. Holland, G. Tully, M. Wilson, DNA commission of the international society for forensic genetics: guidelines for mitochondrial DNA typing, Forensic Sci. Int. 110 (2000) 79–85.
Differences in tissue distribution of HV2 length heteroplasmy in mitochondrial DNA between mothers and children Masaru Asari *, Jun-ichi Azumi, Keiko Shimizu, Hiroshi Shiono Department of Legal Medicine, Asahikawa Medical College, 2-1 Midorigaokahigashi, Asahikawa 078-8510, Japan Received 9 November 2006; received in revised form 15 January 2007; accepted 9 June 2007 Available online 23 July 2007
Abstract Sequence analysis of HV2 in mitochondrial DNA has been performed as a tool for forensic identification, in addition to that of HV1. HV2 contains length heteroplasmy, which shows high variability within an individual or in maternal relatives. In this study, we used cloning analysis and PCR direct sequencing to compare, between mothers and their children, HV2 length heteroplasmic profiles in different tissues. For two mother– child pairs, different types of variant distribution were observed by cloning analysis. In pair 1, length heteroplasmic patterns in most tissues were similar (predominantly 9 and 10Cs variants), but different length heteroplasmic levels, with shifts in predominant genotype, were observed for some hairs in both mother and child. In pair 2, genotype distribution was similar for all tissues, with a predominant 8Cs genotype, but varying in the proportion of minor component. The proportion of one minor length variant (9Cs) in blood from the child was significantly higher than that from the mother, but the proportions of minor components (7 and/or 9Cs) in other tissue samples decreased from mother to child. Moreover, we could confirm that sequence types of PCR products were reflected by the distribution of length variants, which were observed especially in high proportion, in cloning analysis. Our results reveal variable changes in length heteroplasmic level in various tissues between generations. Variability between tissues, especially among hairs, within an individual would result in complicated differences in genotype distribution between maternal generations, and correlate with longer length of Cs for predominant variants. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Mitochondrial DNA; Hypervariable region 2; Length heteroplasmy; Tissue distribution
1. Introduction Sequence analysis of hypervariable regions (HV1, 2 and 3) of mitochondrial DNA (mtDNA), which takes advantage of high copy number per cell, maternal inheritance, lack of recombination and high mutation rate, has been routinely performed in forensic identification for a number of years. In these regions, some kinds of length heteroplasmies, which occur by replication slippage and consist of homopolymeric C-tract or CA repeats, are located at the positions 16180–16193 for HV1 [1], 303–315 for HV2 [2], 514–523 and 568–573 for HV3 [3,4]. HV1 and HV2 length heteroplasmic profiles have been analyzed in detail. The HV1 length heteroplasmy contains a homopolymeric cytosine tract caused by a thymine to cytosine transition at position 16189. However, while this HV1 length heteroplasmy
* Corresponding author. Tel.: +81 166 68 2433; fax: +81 166 68 2439. E-mail address: [email protected] (M. Asari). 0379-0738/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2007.06.015
has been observed, it remains difficult to determine sequence type (e.g., the number of cytosines) in this area by PCR direct sequencing, with the exception of the number of adenines preceding the polycytosine [5]. The HV2 length heteroplasmy is at nucleotide positions 303–315 and consists of seven cytosines followed by a thymine followed by five more cytosines, but five cytosines in the second C-stretch are rarely identified, with six cytosines in this region usually being observed. The HV2 length heteroplasmy is classified into two different types, the first is occurred by cytosine insertion/deletion(s) at the positions 303– 309, and the second caused by a thymine to cytosine transition at position 310 [1,6,7]. Although the former type is frequently observed, the latter is quite rare, thus far observed in only 3 out of 120 Europeans [7]. In cases where the proportion of minor cytosine length variants is much lower than that of a major variant, a predominant HV2 sequence type at 303–315 can be identified by direct sequencing analysis [7]. HV1 length heteroplasmic profiles show wide variation in unrelated individuals [8], but seem to be maintained within
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each lineage [9,10] and in various tissues within individuals [7,11]. On the other hand, instability of the HV2 length heteroplasmy has been reported between one or a few generations [12–14]. In particular, stability dramatically decreases when a predominant genotype contains more than eight cytosines in the first C-stretch [9]. Furthermore, different genotype patterns have been observed between and within tissues [15,16]. Consequently, HV2 length heteroplasmy can be intensively complicated and may be difficult to interpret in different tissues of maternal lineages. In recent years, fragment analyses based on PCR using fluorescently-labeled primers have been introduced as highaccuracy forensic techniques [7,16,17]. Although cloning analysis is a cost and time-consuming method and may not be suitable for application in routine forensic cases, it can reveal the components of length variants and their proportions in detail. Consequently, we used cloning analysis and PCR direct sequencing here to examine HV2 length heteroplasmic profiles between mothers and their children, in order to investigate intra- and intergenerational profile differences.
and 2.5 U Pfu DNA polymerase in 1 PCR buffer (Stratagene, Gebouw, CA). Amplification was conducted in a TaKaRa PCR Thermal Cycler Personal (TaKaRa, Otsu, Japan) using the following conditions: (1) denaturation at 95 8C for 2 min, (2) amplification for 32 cycles at 94 8C for 1 min, 55 8C for 1 min, and 72 8C for 1 min, and (3) final extension at 72 8C for 7 min. PCR products were purified by MinElute PCR Amplification Kit (QIAGEN) to remove unconsumed primers and dNTPs.
2. Materials and methods
3. Results and discussion
2.1. Samples and DNA extraction
In this study, we observed different tissue distributions of HV2 length heteroplasmy between and within two mother– child pairs. To minimize possible PCR errors, each reaction was performed using proof-reading DNA polymerase instead of Taq DNA polymerase [7]. In the cloning analysis, 20 clones obtained by two independently generated PCR products were compared and the results obtained from the different products for each sample showed a quite similar distribution. Length variant distribution based on cloning analysis and sequence types of PCR products for pair 1 are shown in Table 1. By cloning analysis, observed variant types ranged from 7 to 13Cs and 15Cs, and profiles in most tissues, except for some hairs, were apparently heteroplasmic with 9 and 10Cs in high proportion. On the other hand, a slightly heteroplasmic profile, consisting of 9Cs as a major variant and minor length components in varying proportion, was observed in one hair
Peripheral blood, buccal cells, nails and four telogen hairs were collected from each member of two mother–child pairs, all of whom exhibited HV2 length heteroplasmy in blood samples and consisted of a variable number of C residues before T at the position 310. Each hair sample was cut to approximately 1 cm in length including the hair root. DNA extractions from blood and buccal cells were performed by QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany), and from nails and hairs by ISOHAIR (NIPPONGENE, Tokyo, Japan), according to the manufacturer’s instructions. The concentrations of DNA from bloods and buccal cells were determined by Nano Drop ND-1000 (Nano Drop Technologies, Wilmington, DE).
2.2. Amplification Amplification of HV2 was performed using primers L29-H408 [18]. The HV2 segment was amplified in a 25 mL total reaction volume containing 5– 20 ng genomic DNA from bloods and buccal cells or a 5-aliquot of the DNA suspension from nails and hair samples, 0.2 mM of each primer, 200 mM dNTP,
2.3. Cloning PCR products were cloned into pGEM-T Easy Vector (Promega, Madison, WI). Plasmid DNA isolation was performed using QIAprep Spin Miniprep Kit (QIAGEN). For each tissue sample, two independently generated PCR products were cloned and a total of 40 clones (20 clones per PCR product) were used.
2.4. Sequencing Plasmid DNA or purified PCR product was directly sequenced using BigDye Terminator Cycle Sequencing Kit v1.1 (Applied Biosystems, Foster City, CA) according to the manufacturer’s manual. All sequencing reactions were analyzed using the ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Sequences were aligned to the revised Cambridge reference sequence [19].
Table 1 Distribution of length variants and sequence types downstream of position 303 for pair 1 Mother
Variants of Cs 7 8 9 10 11 12 13 15 Sequence type of PCR producta a
Child
Blood
Buccal cells
Nails
Hair1
Hair2
Hair3
Hair4
Blood
Buccal cells
Nails
Hair1
Hair2
Hair3
Hair4
0 1 18 16 4 1 0 0 Ia
0 0 14 22 4 0 0 0 Ia
1 5 13 15 5 1 0 0 Ia
0 3 14 15 3 5 0 0 Ia
1 0 12 15 4 5 1 2 Ia
0 1 19 16 4 0 0 0 Ia
2 5 20 7 3 2 1 0 Ib
0 1 20 18 0 1 0 0 Ia
2 3 15 17 3 0 0 0 Ia
0 2 12 17 9 0 0 0 Ia
0 1 36 3 0 0 0 0 Ic
0 3 33 4 0 0 0 0 Ic
0 0 22 17 1 0 0 0 Ia
0 5 19 13 2 1 0 0 Ia
Categorized into three characteristic types (Type Ia, Ib and Ic). Type Ia does not contain a predominant genotype and shows severely blurred sequences downstream of the first C-stretch within 303–315. Type Ib shows a predominant 9Cs genotype and the sequence downstream of the second C-stretch within 303–315 is unreadable. Type Ic contains a predominant 9Cs genotype and no unreadable sequence.
M. Asari et al. / Forensic Science International 175 (2008) 155–159
Fig. 1. Sequence electropherograms between positions 300 and 320 of representative examples for the three types observed in pair 1. Arrows indicate sites of both T and C peaks observed in equally high proportion.
sample (Hair4) from mother and two hairs (Hair1, 2) from child. Especially in the two hairs from child, very low levels of both the 8 and 10Cs variants were observed (1–4 clones). Corresponding to the distribution of length variants observed in each sample, three characteristic patterns (Type Ia, Ib and Ic) downstream of position 303 were identified by direct sequencing analysis of PCR products. Electropherograms of representative examples for the three types are shown in Fig. 1. In most samples except for Hair4 from the mother and Hairs 1 and 2 from the child, a predominant genotype cannot be identified; what we observed nine unambiguous C peaks followed by both T and C peaks in equally high proportion at the next two consecutive positions
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(Type Ia). The remaining three hair samples commonly showed a predominant 9Cs genotype, with the sequence downstream of the second C-stretch within 303–315 being unreadable in Hair4 from the mother (Type Ib), but identifiable in Hairs 1 and 2 from the child (Type Ic). Although the heteroplasmic patterns for most tissues in pair 1 were quite similar, the strikingly different length heteroplasmic levels observed for some hairs suggest the existence of significant variation among hairs and between tissues within an individual. In general, differences in length heteroplasmic profiles has been explained as a result of a genetic bottleneck due to segregation of the mtDNA, and a larger bottleneck would result in fixed or similar profiles [6,12,16]. For hairs in particular, formation of each hair root and shaft derives from discrete groups of stem cells, compared to other samples such as blood or organ tissues [16,20,21]. Therefore, variable length heteroplasmic levels, with shifts in predominant genotype, could be observed among hairs or across other tissues within an individual, resulting in variability between maternal generations. Furthermore, several studies have shown that differences of point or length heteroplasmy were confirmed not only between hairs and the references (e.g., blood or buccal cells), but also among independent extracts from one hair, both within and between laboratories [22–24]. In addition to the effect of methodology including detection reagents, it has been demonstrated that differential segregation of heteroplasmic variants are observed in various sections of a single hair, and the existence of these differences should be considered in analysis of hair samples. The results for pair 2 are shown in Table 2. By cloning analysis, variant types consisting of 7–11 consecutive Cs were identified, and profiles of all tissues in both mother and child consisted of predominantly 8Cs variants, but varying in the proportion of minor components. As minor components, 7 and 9Cs were identified in blood and hair samples. On the other hand, 9 and 10Cs were commonly observed in buccal cells and nails, and nails from the mother and buccal cells from the child contained 11 and 7Cs, respectively. Furthermore, the profile in one hair (Hair4) from the child is purely homoplasmic, consisting of 8Cs variants alone. With respect to tissue distribution within an
Table 2 Distribution of length variants and sequence types downstream of position 303 for pair 2 Mother
Variants of Cs 7 8 9 10 11 Sequence type of PCR producta
Child
Blood
Buccal cells
Nails
Hair1
Hair2
Hair3
Hair4
Blood
Buccal cells
Nails
Hair1
Hair2
Hair3
Hair4
1 38 1 0 0 IIa
0 32 7 1 0 IIa
0 21 13 5 1 IIc
7 32 1 0 0 IIb
2 36 2 0 0 IIa
8 29 3 0 0 IIb
3 32 5 0 0 IIa
1 31 8 0 0 IIa
1 34 4 1 0 IIa
0 26 12 2 0 IIc
1 39 0 0 0 IIa
0 39 1 0 0 IIa
0 37 3 0 0 IIa
0 40 0 0 0 IIa
a Categorized into three characteristic types (Type IIa, IIb and IIc). Type IIa shows a predominant 8Cs genotype and contains no unreadable sequence in HV2. Type IIb and IIc do not show a predominant genotype, and contain blurred sequence downstream of the first C-stretch within 303–315 and different patterns of multiple T peaks, which interrupt the polycytosine within 303–315. Type IIb contains the pattern with the highest T peak following the second highest T peak, but type IIc contains the pattern with the highest T peak preceding the second highest.
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M. Asari et al. / Forensic Science International 175 (2008) 155–159
individual, the proportions of minor components in nails were significantly higher than in other tissues. Sequences downstream of position 303 for pair 2 by direct sequencing were also classified into three types (Type IIa, IIb and IIc). In most samples, except for nails from both mother and child, and two hairs (Hair1, 3) from mother, C8 genotypes and full-length HV2 sequences were determined (Type IIa). Severely blurred sequence downstream of the first C-stretch was observed in the remaining four samples, and thus a predominant C genotype could not be determined. Moreover, these samples were subdivided into two types based on patterns of multiple T peaks that interrupt the polycytosine within positions 303–315; type IIb contains the pattern with the highest T peak following the second highest T peak, and was observed in nails from both mother and child, but type IIc, which was observed in two hairs (Hair1, 3) from mother, contains the pattern with the highest T peak preceding the second highest. Electropherograms of representative examples for these types are shown in Fig. 2. As in the case of pair 1, we could confirm that the electropherogram for each sample of pair 2 was reflected by the distribution of length variants. As shown by the results for pair 2, a variety of profiles are not necessarily observed among hairs within an individual; profile variety may be associated with longer length of Cs of a predominant genotype in samples such as blood or other organ tissues. However, length-of-Cs genotypes may not strictly correlate with stable or unstable genotype distribution between tissues, including among hairs. In one study, shifts in the
genotype patterns have been observed among hairs in an individual with a 8Cs genotype in blood samples [16]. In terms of maternal distribution in pair 2, the proportion of a minor length variant (9Cs) in blood from the child was significantly higher than that from the mother and the proportions of 7 and/or 9Cs were smaller in the child compared with those of the mother in other tissue samples. These results indicate that different intergenerational shifts of heteroplasmic level across tissues can occur. More complicated qualitative distribution also should be identifiable in various tissues of maternally related members, given that rapid shifts in predominant genotype have been observed between one or a few generations [6,13]. In the forensic field, the existence of heteroplasmy, and differences in the proportion of components between generations or within individuals, has made interpretation of mtDNA sequences more difficult. As shown in the published guidelines [25] and recommendations [26], determination of the number of cytosines is difficult and the exact number should not be considered, if a characteristic ‘‘out-of-phase’’ pattern downstream of the first C-stretch within positions 303–315 is observed. However, profiles lacking the exact number of cytosines sometimes contain enough information to be considered useful for identification purposes. In terms of leading to a decision (inclusive, exclusive or inconclusive), comparison of length heteroplasmic profiles can be valuable and informative. In conclusion, our results reveal variable changes in length heteroplasmic level in various tissues between generations. Variability between tissues, especially among hairs, within an individual would result in complicated differences in genotype distribution between maternal generations, and correlate with longer length of Cs for predominant variants. References
Fig. 2. Sequence electropherograms between positions 300 and 320 of representative examples for the three types observed in pair 2. Arrows indicate sites of both T and C peaks observed in equally high proportion.
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