Biological Significance and Profile of Length Heteroplasmy in the Hypervariable Segments of the Human Mitochondrial DNA Control Regions from Blood Cells.

Abstract A high incidence of mitochondrial DNA (mtDNA) variations was observed in both hypervariable region (HV) 1 and HV2; most mtDNA sequence variations were localized at poly C tract at nucleotides (nt) 303-315 (CCCCCCCTCCCCC, 7CT5C) in the HV2. Another poly C tract variant in HV1 at nt 16184-6193 have been suggested to be related with diabetes, dilated cardiomyopathy and some cancers. Poly C tract in HV2 is part of the conserved sequence block II located in 92-bp from the heavy strand replication origin. It is not yet clear whether poly C variants at nt 303–315 would lead to alterations in mtDNA replication. We hypothesized that some severe alterations in poly C tracts may lead to impairment of mtDNA replication. Here we present the profile of length heteroplasmy in HV from blood cells and its biological significance. A total of 57 maternally unrelated healthy donors were included and heparinized bloods were obtained from five age groups including 12 cord bloods. We amplified and sequenced the 1,121-bp control region including HV1 and HV2. In an attempt to investigate mtDNA length heteroplasmy, we carried out a qualitative and quantitative profiling length heteroplasmy using size-based PCR product separation by capillary electrophoresis (ABI 3100 Genetic Analyzer and ABI Prism Genotyper version 3.1). Length heteroplasmy was further confirmed by cloning and sequencing. Quantitative analysis of mtDNA molecules was performed using the QuantiTect SYBR Green PCR kit (Qiagen) and Rotor-Gene 3000 (Corbett Research) and standard plot was obtained from cloned cytochrome b gene. The mtDNA control region sequences showed 57 different haplotypes resulting from 77 polymorphic positions. Common polymorphisms were 73A>G (98%), 263A>G (91%), 16223C>T (47%), 16189T>C (35%), 150C>T (25%) and 152T>C (18%). The patterns of length heteroplasmy in the HV2 region were classified into 6 types. In the HV1 region, length heteroplasmy showed 8 variant peak patterns. The distribution of length heteroplasmy in poly C tracts at nt 303 – 315 was mtDNA mixture of 7CT6C+8CT6C (53%), 8CT6C+9CT6C (26%), 8CT6C+9CT6C+10CT6C (11%), 9CT6C+10CT6C +11CT6C (5%), 9CT6C+10CT6C (3%) and 7CT6C+6CT6C (2%). The distribution of length heteroplasmy pattern in poly C tract at nt 16184 – 16193 was 5CT4C+5CT3C (60%), 9C+10C+11C+12C (21%), 9C+10C+11C (5%), 3CT6C+3CT5C (3%), 9C+10C+11C+12C+13C (3%), 3CT4C+3CT3C (3%), 10C+11C+12C (2%), and 8C+9C+10C+11C+12C (2%). Interestingly, this study revealed that all healthy subjects showed length heteroplasmy in the HV1 and HV2 regions in contrast to previous studies. Length heteroplasmy in poly C 303–315 showed two groups of two peaks (n = 48) and more than three peaks (n = 9). MtDNA content from group with three peaks in poly C 303–315 (61,983,373 molecules/ul ± 33,219,871, mean±SD) was markedly lower than those with two peaks (133,777,955 molecules/ul ± 87,209,377). In conclusion, significantly higher rate of length heteroplasmy was observed in HV1 and HV2 from healthy donors and the presence of more than three mtDNA types in poly C at nt 303 – 315 might be associated with impairment of mtDNA replication.

Download Full-text

Profound Length Heteroplasmic Alterations in Mitochondrial DNA Control Region From Primary AML Cells: Implication of Impaired Mitochondrial Replication and Demonstration of ‘vicious cycle’ Hypothesis.

Blood ◽

10.1182/blood.v114.22.3116.3116 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3116-3116

Author(s):

Myung-Geun Shin ◽

Hye Ran Kim ◽

Hyeoung-Joon Kim ◽

Hoon Kook ◽

Tai Ju Hwang ◽

...

Keyword(s):

Mitochondrial Dna ◽

Control Region ◽

Copy Number ◽

Regulatory Region ◽

Mtdna Control Region ◽

Vicious Cycle ◽

Mtdna Copy Number ◽

Length Heteroplasmy ◽

Region Length ◽

D Loop

Abstract Abstract 3116 Poster Board III-53 Mitochondrial DNA (mtDNA) control region (displacement (D)-loop including HV1 and HV2) is a non-coding region of 1124 bp (nucleotide positions, np 16 024–576), which acts as a promoter for both the heavy and light strands of mtDNA, and contains essential transcription and replication elements (Blood 2004;103:4466-77). Importantly, mutations in the D-loop regulatory region might change mtDNA replication rate by modifying the binding affinity of significant trans-activating factors (Eur J Cancer 2004;40:2519-24). Thus, length heteroplasmic alterations of mtDNA control region may be related with mitochondrial dysfunction resulting in ‘vicious cycle’ (Mol Med Today 2000;6:425-32). In an attempt to investigate profiling of mtDNA length heteroplasmic alterations in primary AML cells, we carried out a quantitative size-based PCR product separation by capillary electrophoresis (ABI 3130XL Genetic Analyzer and ABI Prism Genotyper version 3.1) using six targets (np 303-315 poly C, np 16184-16193 poly C, np 514-511 CA repeats, np 3566-3572 poly C, np 12385-12391 poly C and np 12418-12426 poly A). Length heteroplasmy was further confirmed by cloning and sequencing. Quantitative analysis of mtDNA molecules was performed using the QuantiTect SYBR Green PCR kit (Qiagen) and Rotor-Gene 3000 (Corbett Research). Forty-eight AML bone marrow samples were collected after receiving Institutional Review Board approval and informed consent. There were profound alterations of mtGI in 303 poly C, 16184 poly C and 514 CA repeats. The length heteroplasmy pattern of 303 poly C tract in the HV2 region disclosed mixture of 7C, 8C, 9C and 10C mtDNA types. In the HV2 region, length heteroplasmy in poly-C tract at np 303 - 309 exhibited 5 variant peak patterns: 7CT6C+8CT6C (50.0%), 8CT6C+9CT6C (14.0%), 8CT6C+ 9CT6C+ 10CT6C (10.4%), 9CT6C+10CT6C+11CT6C (8.3%) 9CT6C + 10CT6C + 11CT6C+12CT6C (2.1%). The length heteroplasmy pattern of 514-523 CA repeats in the HV2 region exhibited 2 variant peak patterns: CACACACACA (56.3%) and CACACACA (43.7%). In the HV1 region, length heteroplasmy in the poly-C tract at np 16184 - 16193 exhibited 9 variant peak patterns: 5CT4C+5CT3C (31.0%), 6CT4C+6CT3C (2.1%), 9C+10C+11C+12C (16.7%), 9C+10C+11C (2.1%), T4CT4C+5CT3C (4.2%), 9C+10C+11C+12C+13C (2.1%), 3CTC4C+5CT3C (2.1%), 10C+11C+12C+13C (4.2%), 8C+9C+10+11C (2.1%). Primary AML cells revealed decreased enzyme activity in respiratory chain complex I, II and III. AML cells had about a two-fold decrease in mtDNA copy number compared with normal blood mononuclear cells. Current study demonstrates that profound length heteroplasmic alterations in mtDNA control region of primary AML cells may lead to impairment of mitochondrial biogenesis (reduction of mtDNA copy number) and derangement of mitochondrial ATP synthesis. During this perturbation, mitochondria in primary AML cells might produce a large amount of reactive oxygen species, which causes the vicious cycle observed in chronic inflammatory diseases and cancers as well. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Evaluating length heteroplasmy in the human mitochondrial DNA control region

International Journal of Legal Medicine ◽

10.1007/s00414-009-0385-0 ◽

2009 ◽

Vol 124 (2) ◽

pp. 133-142 ◽

Cited By ~ 21

Author(s):

Lucy Forster ◽

Peter Forster ◽

Susan M. R. Gurney ◽

Matthew Spencer ◽

Christopher Huang ◽

...

Keyword(s):

Mitochondrial Dna ◽

Control Region ◽

Length Heteroplasmy ◽

Mitochondrial Dna Control Region ◽

Human Mitochondrial Dna

Download Full-text

Erratum to: Evaluating length heteroplasmy in the human mitochondrial DNA control region

International Journal of Legal Medicine ◽

10.1007/s00414-010-0435-7 ◽

2010 ◽

Vol 124 (4) ◽

pp. 319-319

Author(s):

Lucy Forster ◽

Peter Forster ◽

Susan M. R. Gurney ◽

Matthew Spencer ◽

Christopher Huang ◽

...

Keyword(s):

Mitochondrial Dna ◽

Control Region ◽

Length Heteroplasmy ◽

Mitochondrial Dna Control Region ◽

Human Mitochondrial Dna

Download Full-text

Investigation of length heteroplasmy in mitochondrial DNA control region by massively parallel sequencing

Forensic Science International Genetics ◽

10.1016/j.fsigen.2017.07.003 ◽

2017 ◽

Vol 30 ◽

pp. 127-133 ◽

Cited By ~ 3

Author(s):

Chun-Yen Lin ◽

Li-Chin Tsai ◽

Hsing-Mei Hsieh ◽

Chia-Hung Huang ◽

Yu-Jen Yu ◽

...

Keyword(s):

Mitochondrial Dna ◽

Control Region ◽

Massively Parallel Sequencing ◽

Massively Parallel ◽

Length Heteroplasmy ◽

Mitochondrial Dna Control Region ◽

Parallel Sequencing

Download Full-text

Length heteroplasmy in the predominate mitochondrial DNA haplogroups in the Croatian population

Molecular and experimental biology in medicine ◽

10.33602/mebm.2.1.3 ◽

2019 ◽

Vol 2 (1) ◽

pp. 19-23

Author(s):

Lucija Barbaric ◽

Korana Lipovac ◽

Viktorija Sukser ◽

Sara Rozic ◽

Marina Korolija

Keyword(s):

Mitochondrial Dna ◽

Mitochondrial Genome ◽

Control Region ◽

Mitochondrial Control Region ◽

Length Heteroplasmy ◽

Data Support ◽

Variable Segment

Mitochondrial control region represents the most variable segment of the mitochondrial genome. The frequency and pattern of heteroplasmy has been described in several studies; however, none of the reports documented the Croatian population. In the present study, we screened the control region (1122 bp) of 95 individuals belonging to two predominant mitochondrial phylogenetic branches in the Croatian population, haplogroups H and U. Length heteroplasmy occurred in polycytosine (poly-C) tracts within three hypervariable segments of the control region with the following frequencies: HVSI - 26.3%, HVSII - 52.6% and HVSIII - 7.4%. Furthermore, the association between certain polymorphisms in HVSI and length heteroplasmy was investigated. Our results indicate that only polymorphisms located in the poly-C tract are associated with HVSI length heteroplasmy. The T to C transition at np 16189 is significantly associated with the occurrence of length heteroplasmy (p<0.0001). This effect was even stronger if the C insertion was present in the position 16193. The data support the hypothesis that an uninterrupted poly-C tract of more than eight cytosines leads to length heteroplasmy. Length heteroplasmy associated with the T to C substitution in np 16189 was predominantly found in haplogroup U.

Download Full-text

Sequence variations in the mitochondrial DNA control region and their implications for the phylogeny of the Cypriniformes

Canadian Journal of Zoology ◽

10.1139/z02-035 ◽

2002 ◽

Vol 80 (3) ◽

pp. 569-581 ◽

Cited By ~ 56

Author(s):

Huanzhang Liu ◽

Chyng-Shyan Tzeng ◽

Hui-Yu Teng

Keyword(s):

Phylogenetic Analysis ◽

Mitochondrial Dna ◽

Control Region ◽

Monophyletic Group ◽

Mitochondrial Dna Control Region ◽

Central Domain ◽

Conserved Sequence ◽

Sequence Block ◽

Sequence Variations

The mitochondrial DNA control region of six cobitids and two catostomids was sequenced and compared with sequences of other cypriniforms to study their sequence variations. The extended termination associated sequence (ETAS) domain, central domain, and conserved sequence block (CSB) domain were partitioned and the ETAS sequence, CSB-D, CSB-E, ECSB-F, CSB1, CSB2, and CSB3 were identified. It is suggested that the "hairpin" TACAT-ATGTA is the key sequence of ETAS and GACATA is the symbol of CSB1. Phylogenetic analysis based on the CSB domain showed that all cyprinids evolved as one monophyletic group, while the non-cyprinid Cypriniformes could be another monophyly that is in accordance with the hypothesis proposed by Siebert. Further analysis of the phylogeny of the Cobitoidei was also conducted and it is tentatively suggested that their relationships are Catostomidae + (Gyrinocheilidae + (Botiinae + (Homalopteridae + (Cobitinae + Nemacheilinae)))).

Download Full-text

Tandemly repeated sequences in mtDNA control region of whitefish, Coregonus lavaretus

Genome ◽

10.1139/g00-001 ◽

2000 ◽

Vol 43 (3) ◽

pp. 584-587 ◽

Cited By ~ 11

Author(s):

Pawel Brzuzan

Keyword(s):

Mitochondrial Dna ◽

Control Region ◽

Pcr Amplification ◽

Repeated Sequences ◽

Coregonus Lavaretus ◽

Length Variation ◽

Conserved Sequence ◽

Repeat Units ◽

Pcr Products ◽

D Loop

Length variation of the mitochondrial DNA control region was observed with PCR amplification of a sample of 138 whitefish (Coregonus lavaretus). Nucleotide sequences of representative PCR products showed that the variation was due to the presence of an approximately 100-bp motif tandemly repeated two, three, or five times in the region between the conserved sequence block-3 (CSB-3) and the gene for phenylalanine tRNA. This is the first report on the tandem array composed of long repeat units in mitochondrial DNA of salmonids.Key words: Coregonus lavaretus, D-loop mitochondrial DNA, tandemly repeated sequences.

Download Full-text