Biological Significance and Profile of Length Heteroplasmy in the Hypervariable Segments of the Human Mitochondrial DNA Control Regions from Blood Cells.
Authors: Shin Myung-Geun , Kim Hyeoung-Joon , Kim Hye-Ran , Kim Hee-Nam , Lee Il-Kwon , Kook Hoon , Cho Duck , Shin Jong-Hee , Suh Soon-Pal , Ryang Dong-Wook 
Published: October 2019

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.