TROPICAL GENETICS
Volume 1.
No 1.
May, 2021 https://ojs.
genetikawan-muda.
com/index.
php/tg Original Research Identification of single nucleotide polymorphisms on the D-loop region of mtDNA in Sundanese population Wolly Candramila1*.
Sony Heru Sumarsono2.
Bambang Suryobroto3.
Maelita Ramdani Moeis2 1Department of Biology Education.
Faculty of Teacher Training and Education.
Universitas Tanjungpura.
Pontianak.
Indonesia, 78124 School of Life Sciences and Technology.
Institut Teknologi Bandung.
Bandung.
Indonesia, 40132 3Department of Biology.
Faculty of Mathematics and Natural Sciences.
Institut Pertanian Bogor University.
Bogor.
Indonesia, 16680 *corresponding author E-mail address: wolly.
candramila@fkip.
Article Info Abstract Article history:
Received 30 January 2021 Received in revised form 4 March 2021 Accepted 2 April 2021 Available online 30 May 2021 Identification of sequence polymorphism on the D-loop region of mtDNA has been done for various purposes, including health and medical treatment.
In this research, single nucleotide polymorphisms were identified in the D-loop region of mtDNA of the Sundanese population in western Java.
A total of 118 unrelated and healthy Sundanese probands were collected from closed-traditional kampung adat and open communities distributed in 14 cities and regencies in western Java.
DNA amplification and direct sequencing of the D-loop region was proceeded using primers L15990 and H409.
Multi-alignment was conducted not only intrapopulation but also with D-loop sequence data stored in GenBank for comparison.
In this research, we categorized high frequency SNPs as less effective for identification in population studies because of their present in other population outside Indonesia.
Meanwhile, lower-frequency SNPs showed typical variants of Sundanese haplotypes.
On the other hand, rare or low-frequency SNPs should be re-examined in larger size of samples to have better understanding about risk factor for many diseases.
Keywords:
SNPs D-loop region Sundanese Multi-alignment mtDNA How to cite:
Candramila.
Sumarsono.
Suryobroto.
Moeis.
Identification of single nucleotide polymorphisms on the D-loop region of mtDNA in Sundanese population.
Tropical Genetics 1.
: 17-23.
Copyright A 2021.
The Authors.
This is an open access article under the CC BY-NC-SA license .
ttp://creativecommons.
org/licenses/by-nc-sa/4.
0/).
Introduction many diseases, such as cancers (Guo et al.
Kong et al.
, 2.
and chronic kidney failure (Xu et al.
, 2.
Murakami et al.
also found that polymorphism on the D-loop region might cause different durability in training among individuals.
However, there is still limited information about single nucleotide polymorphisms (SNP.
on the Dloop region among populations in Indonesia when compared to more than 1300 ethnics encompassing the whole areas of the The Sundanese people are the secondlargest ethnic in Indonesia mainly distributed in western Java (Badan Pusat Statistik, 2.
Migration and mixed marriage processes The hypervariable (HV) I and II regions that lie on the D-loop region of the mitochondrial DNA are conventionally used for determining the identity in population studies (Budowle et , 2002.
Sharma et al.
, 2005.
Tuladhar et al.
Ranasinghe et al.
, 2.
and forensic purposes (Andryasson et al.
, 2002.
Coble et , 2.
In progress, databases of single nucleotide polymorphism (SNP) on the Dloop region of mtDNA are continuously being identified in many other populations and for other purposes.
Studies showed that the sequence polymorphisms on the D-loop regions were significantly associated with Candramila.
et al.
Tropical Genetics 1.
: 17-23 urged the people to be distributed to many other regions in Indonesia.
Being highly distributed to many regions and its maternal inheritability through mixed marriage process may also cause these specific SNPs found in the Sundanese population can be exhibited in other cross-marriage ethnic and vice versa.
More information about these SNPs will help better understanding about potential genetic identity and its changing through mutation In this research, we identified SNPs of the HVI and HVII on the D-loop regions of mtDNA among Sundanese population in western Java.
reaction method using L15990 .
AoTTAACTCCACCATTAGCACC-3A.
and H409 .
AoCTGTTaGTGCATACCGCC-3A.
as forward and reverse primers, subsequently.
The numbers following the L and H chains denote the 3Ao end of the primer in accordance to the mtDNA sequence of Anderson et al.
and Andrews et al.
Primer H409 was previously used by Siguryardyttir et al.
, meanwhile.
L15990 was originally designed based on the nucleotide specificity found in the blasting result of the D-loop Primers were synthesized in such specific orders by Integrated DNA Technologies.
Singapore.
The amplification reaction was following the product protocol for DreamTaq Green PCR Master Mix .
X) from Thermo Scientific (Thermo Fisher Scientific.
Inc.
Initial denaturation was set at 95AC for 3 min, followed by another denaturation at 95AC for 30 seconds, primer annealing at 54-58AC for 30 s, and elongation step at 72AC for 1 min and 15 s.
The whole cycle was repeated 30-35 times and followed by final elongation at 72AC for 10 min.
DNA
amplification was using T100TM Thermal Cycler (Bio-Rad Laboratories.
Inc.
USA).
The PCR product was visualized on 1% agarose gel soaked in 0.
5 AAg/ml EtBr solution for 15 min.
Materials and Methods Probands criteria and ethical clearance A total of 118 unrelated Sundanese probands were collected from two types of settlement areas in West Java Province, including random open communities and seven closed-traditional kampung adat.
Probands distributed in 14 cities and regencies in western Java.
ParentAos origin and pure Sundanese ethnicity were checked and those who fully understood the informed consent and volunteered freely were included in the Ethical clearance (No.
222/FKUPRSHS/KEPK/Kep.
/EC/2.
for the whole activities of the research was released by the Faculty of Medicine.
Padjadjaran University & RSUP dr.
Hassan Sadikin in Bandung.
DNA Sequencing The PCR products were purified using Xprep Gel & PCR Purification Kit (PhileKorea Technology.
Inc.
Kore.
The direct sequencing method of Sanger et al.
with the same primer designs as amplification was used to read the amplified DNA.
The bidirectional sequencing protocols were done at Macrogen.
Inc.
South Korea.
DNA Collection Whole DNA samples were extracted from blood as described in Sambrook et al.
Blood collection was conducted from March 2011 until March 2014.
Extracted DNA were added with 200 AAl of elution buffer, left for 5 min until fine diluted, and then stored at 20AC for further use.
Data Analysis Sequencing data in the chromatogram and the nucleotide orders were analyzed in DNA
Baser v4 (DNA Baser Sequence Assembler
Heracle Biosoft
SRL,
DnaBaser.
Only samples with nucleotide reading peaks characterized by QVO 22 would be further analyzed.
Multiple DNA Amplification The amplification of the D-loop region of mtDNA was done by polymerase chain Candramila.
et al.
Tropical Genetics 1.
: 17-23 alignments were done with MUSCLE 3.
(Edgar, 2.
in MEGA5.
2 (Tamura et al.
The alignment results were recorrected manually in TextPad format.
phylogenetic alignment steps by Bandelt and Parson .
as a review of the recommendation by (Wilson et al.
, 2002a,.
Sequence polymorphisms were identified both in HVI and HVII in the D-loop region of mtDNA.
The determination of mutation types was done by comparing the samples to the homolog D-loop sequence of rCRS .
evised Cambridge Reference Sequence.
NC_012.
by Andrews et al.
The comparison was also done with other homolog sequences GenBank .
ttp://w.
gov/genbank/) notably for Asian (Japan.
AF346989 and AF346990.
Mongolia/China.
AY255146 and JN857056.
India.
FJ383814 and AY713976.
and Thailand.
FJ442939 and GU810.
East European (JF937.
Papua New Guinean (AY289092 and AY289.
, and African (D38.
Mutations found in only one individual are not counted.
Results and Discussion D-loop sequence polymorphism was analysed on position 16180 until 365 of the circular mtDNA with a total length of 824 base The sequence analysis was grouped into two positions of HVI and HVII regions.
found 181 variants and 68 multiallelic positions following alignment of the raw sequence data.
After rejecting variants found in only one individual, the types of mutation in HVI and HVII regions were confirmed for 79 and 91 SNPs, respectively (Table .
Sequence polymorphism per base were slightly higher in HVII .
than HVI .
Transitions .
8%) are more frequent than other types of mutation .
1%).
As found in coding regions, transitions are favoured several folds over transversion but the evolutionary basis for conservative protein selection is not large enough (Stoltzfus and Norris, 2.
Table 1.
Target regions used in this study and the number of SNPs.
Number of Region
SNPs per Region Base position* SNPs
Size
HVI
HVII
Multiallelic SNPs
TS*
TV*
Indel* *Base position according to Andrews et al.
TS: Transition.
TV: Transversion.
Indel: Insertion/Deletion Total types of base mutation encountered on the HVI and HVII are shown in Table 2.
Transition T16519C .
%) was most frequent among all samples in HVI region, meanwhile, 3C .
%), 315.
1C .
%), and transition A73G .
%) are most frequent than other mutations in HVII region.
Mutations T16519C, 315.
1C, and A73G were also found in other compared populations stored in GenBank, however, insertion 302.
3C was only found in Papua New Guinean and East European sequence data.
These SNPs are assumed to be less effective for population Table 2.
Types of base mutation on HVI and HVII regions of mtDNA identified in this study.
Region & Region & Freq.
Position Type of mutation Position Type of mutation (%) HVI
T16519C
Transition HVII .
A73G Transition .
92C16223T Transition .
315,1C
Insertion
T16209C
Transition 302,3C Insertion
377 bp
T16304C
Transition T152C Transition C16261T Transition T146C Transition T16217C Transition C64T Transition A16235G Transition C150T Transition A16272G Transition G316A Transition Freq.
(%)
Candramila.
et al.
C16266A
T16311C
T16362C
C16234T
C16257A
C16358T
C16292T
G16558A
G16213A
C16294T
G16434A
G16384A
C16290T
C16355T
C16444T
C16501T
C16197del
A16293C
G16390A
A16402C
C16221T
A16258G
T16263C
T16297C
T16298C
C16431A
C16192T
C16197T
T16198C
C16228A
A16258C
T16276A
C16278T
A16326C
C16327T
A16343T
T16386A
T16437G
C16451T
G16496A
C16560A
T16568G
C16193T
C16197G
G16204A
T16224C
C16228T
A16241G
C16242T
C16248A
G16255A
A16265C
C16266T
C16270T
G16274A
C16279A
A16293G
C16301A
G16319A
A16335G
T16381del Tropical Genetics 1.
: 17-23
Transversion Transition Transition Transition Transversion Transition Transition Transition Transition Transition Transition Transition Transition Transition Transition Transition Deletion Transversion Transition Transversion Transition Transition Transition Transition Transition Transversion Transition Transition Transition Transversion Transversion Transversion Transition Transition Transition Transversion Transversion Transversion Transition Transition Transversion Transversion Transition Transversion Transition Transition Transition Transition Transition Transversion Transition Transversion Transition Transition Transition Transversion Transition Transversion Transition Transition Deletion A210G
G225A
T72G
302,2C
309,1C
A111C
C186G
356,3A
G275A
A297C
C61T
T74G
T195C
T318C
C6A
A16C
G81A
C91G
G94A
C132T
C151T
G207A
C258A
A278T
T55G
A200G
T204G
C253T
C295T
C343T
A28C
G92A
G103C
A160T
A165C
A183G
T199C
A257C
A278del
C298A
302,2A
C320T
C324G
356,3C
T42C
G62del
A77C
G79C
G109A
G124A
T125A
T131G
C132del
T142C
G143A
A153G
A181C
A183del
G187A
C190A
T199A
Transition Transition Transversion Insertion Insertion Transversion Transversion Insertion Transition Transversion Transition Transversion Transition Transition Transversion Transversion Transition Transversion Transition Transition Transition Transition Transversion Transversion Transversion Transition Transversion Transition Transition Transition Transversion Transition Transversion Transversion Transversion Transition Transition Transversion Deletion Transversion Insertion Transition Transversion Insertion Transition Deletion Transversion Transversion Transition Transition Transversion Transversion Deletion Transition Transition Transition Transversion Deletion Transition Transversion Transversion Candramila.
et al.
G16398A
C16467T
T16469G
A16492T
C16498A
T16502G
G16526C
C16542T
T16555A
A16564C
Tropical Genetics 1.
: 17-23
Transition Transition Transversion Transversion Transition Transversion Transversion Transition Transversion Transversion T199del
T206G
T209A
T220G
C222G
A227G
T236A
C242A
C264T
269,1A
A270C
A272C
C273A
C285A
C299A
302,1C
302,3A
309,1T
T310C
315,1G
A341T
C353A
The types of mutation with lower frequency were found in transitions C16223T %).
T152C .
%).
T16209C .
%).
T16304C
%), and C16261T .
%).
Transition C16223T and T152C were more likely found in Asian populations, namely Japanese.
Chinese.
Thailand as well as in Taiwanese .
%) (Tsai et al.
, 2.
, meanwhile, transition C16261T was also found in Papua New Guinean and India.
On the other hand, transitions T16209C and T16304 were only found in Sundanese.
These two mutations were encountered in some of the common haplotypes found in the Sundanese population, namely Haplotypes A.
C, and D for transition T16304C and Haplotypes H.
J, and K for transition T16209C (Candramila et al.
, 2.
Other types of mutation with lower frequency were transitions T146C .
%).
T16217C .
%).
C64T .
%).
C150T .
%).
A16235G .
%).
A16272G .
%).
G316A
%).
A210G .
%).
G225A .
%).
T16311C
%), and T16362C .
%), and transversions C16266A .
%) and T72G .
%).
Mutations A16235G.
A16272G.
G316A.
A210G, and G225A were only found in Sundanese As reported by Candramila et al.
, transition A16235G was common for Haplotype K.
A210G for Haplotype J, and A16272G.
G316A and G225A for Haplotype Deletion Transversion Transversion Transversion Transversion Transition Transversion Transversion Transition Insertion Transversion Transversion Transversion Transversion Transversion Insertion Insertion Insertion Transition Insertion Transversion Transversion A1.
Meanwhile, transition T146C was common in Haplotype A2 of the Sundanese population but this mutation was also found in Papua New Guinean (AY289.
Thailand (FJ442.
, as well as in Taiwanese Han population (Tsai et al.
, 2.
Most SNPs identified in this study were found in less than 10% of samples.
The analysis of low-frequency SNPs is valuable in determining the risk factor for common diseases (Kryukov et al.
, 2.
Babron et al.
found that different frequency categories showed different stratification D-loop sequence polymorphisms were also reported for their association with various diseases.
For example.
Xu et al.
reported six SNPs .
G, 146C, 150T, 194T, 195C and 310C) as risk factors for chronic kidney disease, meanwhile.
Govatati et al.
showed that 189G/310TC/16189C haplotype may be associated with an increased risk for endometriosis.
Moreover, polymorphisms at C16069T.
T16126C.
T16189C.
T16519C and C16223T were correlated with an increased risk of Huntington Disease (HD) while SNPs at C16150T.
T16086C and T16195C were associated with a decreased risk of Huntington's disease (Mousavizadeh et al.
Most of the polymorphisms associated Candramila.
et al.
Tropical Genetics 1.
: 17-23 Pyrosequencing Technology.
Biotechniques, 32.
, 124-133.
Andrews.
Kubacka.
Chinnery.
Lightowlers.
Tumbull.
, & Howell.
Reanalysis and Revision of the Cambridge Reference Sequence fo Human Mitochondrial DNA.
Nature Genetics, 23, 147.
Babron.
-C.
, de Tayrac.
Rutledge.
Zeggini.
, & Genin.
Rare and Low Frequency Variant Stratification in the UK Population: Description and Impact on Association Tests.
PLoS ONE, 7.
, e46519.
https://doi.
org/10.
1371/journal.
Badan Pusat Statistik.
Kewarganegaraan.
Suku Bangsa.
Agama, dan Bahasa Sehari-hari Penduduk Indonesia.
Jakarta: Badan Pusat Statistik.
Bandelt.
-J.
, & Parson.
Consistent treatment of length variants in the human mtDNA control region: a reappraisal.
International Journal of Legal Medicine, 122.
Budowle.
Allard.
Fisher.
Isenberg, .
Monson.
Stewart.
Miller.
HVI and HVII mitochondrial DNA data in Apaches and Navajos.
International Journal of Legal Medicine, https://doi.
org/10.
1007/s00414-001-0283-6.
Candramila.
Sumarsono.
Suryobroto.
& Moeis.
Maternal Genetic Distance Between Sundanese and Javanese Populations in Indonesia.
EPiC Series in Biological Sciences .
Manchester:
Easy Chair.
Coble.
Vallone.
Just.
Diegoli.
Smith.
, & Parsons.
Effective Strategies for Forensic Analysis in the Mitochondrial DNA Coding Region.
International Journal of Legal Medicine, 120, https://doi.
org/10.
1007/s00414-0050044-z.
Edgar.
MUSCLE: a multiple sequence alignment method with reduced time and space complexity.
BMC Bioinformatics, 5, 1-19.
Fucharoen.
Fucharoen.
, & Horai.
Mitochondrial DNA Polymorphisms in Thailand.
Journal of Human Genetics, 46, 115125.
Govatati.
Deenadayal.
Shivaji.
, &
Bhanoori.
Mitochondrial displacement loop alterations are associated with endometriosis.
Fertility and Sterility, 99.
, with those diseases were also found in Sundanese samples, however, more ongoing research with a large sample size and functional evaluation of identified SNPs are needed to validate the findings.
Conclusions We identified 170 sequence nucleotide polymorphisms and 14 multiallelic positions in HV I and II regions of the mtDNA.
Highfrequency SNPs (T16519C 87%, 302.
3C 57%,
1C 98%, and A73G 99%) were also found in populations outside Indonesia, therefore, less effective for identification in population Meanwhile, lower-frequency SNPs were more common for Sundanese haplotypes (T16304C 24%.
T16209C 25%.
A16235G 15%.
A210G 14%.
A16272G 15%.
G316A 15%.
G225A 14%, and T146C 19%).
the other hand, rare or low-frequency SNPs may be associated with risk factor for many diseases and failures, but more studies are needed to support the findings in Sundanese Acknowledgments The authors thank Ministry of Research.
Technology, and Higher Education for the fund of Doctoral Research Program Year 2012 and the governmental offices in West Java Province for the permit of the research in all sampling locations, as well as all participants to make this research came to reality.
Conflict of Interest None.
References Anderson.
Bankier.
Barrel.
, de Bruijn, .
Coulson.
Drouin.
Young.
Sequence and the Organization of the Human Mitochondrial Genome.
Nature, 209.
Andryasson.
Asp.
Alderborn.
Gyllensten, , & Allen.
Mitochondrial Sequence Analysis for Forensic Identification Using Candramila.
et al.
Tropical Genetics 1.
: 17-23 https://doi.
org/10.
1016/j.
Guo.
Zhao.
Fan.
Du.
Zhao.
, & Wang.
Identification of Sequence Polymorphisms in the D-loop Region of Mitochondrial DNA as a Risk Factor for Colon Cancer.
Mitochondrial DNA Part A, 1-2.
Kong.
Shi.
, & Li.
Single Nucleotide Polymorphisms in the D-loop Region of Mitochondrial DNA are Associated with Epithelial Ovarian Cancer Prognosis.
Mitochondrial DNA Part A, 1-3.
Kryukov.
Pennacchio.
, & Sunyaev.
Most rare missense alleles are deleterious in humans: implications for complex disease and association studies.
American Journal of Human Genetics, 80.
, https://doi.
org/10.
1085/513473.
Mousavizadeh.
Rajabi.
Alaee.
Dadgar.
& Houshmand.
Usage of mitochondrial D-loop variation to predict risk for Huntington disease.
Mitochondrial DNA, 26.
, https://doi.
org/10.
3109/19401736.
Murakami.
Ota.
Simojo.
Okada.
Ajisaka.
, & Kuno.
Polymorphisms in Control Region of mtDNA Relates to Individual Differences in Endurance Capacity or Trainability.
Japanese Journal of Physiology, 52.
, 247-256.
Nishimaki.
Sato.
Fang.
Ma.
Hasekura, , & Boetrcher.
Sequence polymorphism in the mtDNA HV1 Region in japanese and Chinese.
Legal Medicine, 1, 238249.
Piercy.
Sullivan.
Benson.
, & Gill.
The application of mitochondrial DNA typing to the study of white Caucasian genetic International Journal of Legal Medicine, 106, 85-90.
Ranasinghe.
Tennekoon.
Karunanayake, .
Lembring.
, & Allen.
A study of genetic polymorphisms in mitochondrial DNA hypervariable regions I and II of the five major ethnic groups and Vedda population in Sri Langka.
Legal Medicine, 17.
, 539-546.
https://doi.
org/10.
1016/j.
Rousselet.
, & Mangin.
Mitochondrial DNA Polymorphisms: a study of 50 French Caucasian individuals and application to forensic casework.
International Journal of Legal Medicine, 111, 292-298.
Sambrook.
Fritsch.
, & Maniatis.
Molecular Cloning: A Laboratory Manual 2nd Edition.
New York: Cold Spring Harbor Laboratory Press.
Sharma.
Saha.
Rai.
Bhat.
, & Bamezai.
Human mtDNA hypervariable regions.
HVR I and II, hint at deep common maternal founder and subsequent maternal gene flow in Indian population groups.
Journal of Human Genetics, https://doi.
org/10.
1007/s10038-005-0284-2.
Siguryardyttir.
Helgason.
Gulcher.
Stefansson.
, & Donnelly.
The Mutation Rate in the Human mtDNA Control Region.
American Journal of Human Genetics, 66, 15991609.
Stoltzfus.
, & Norris.
On the causes of Evolutionary Transition:Transversion Bias.
Molecular Biology and Evolution, 33.
, 595602.
https://doi.
org/10.
1093/molbev/msv274.
Tamura.
Peterson.
Peterson.
Stecher.
Nei.
, & Kumar.
Mega5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood.
Evolutionary Distance, and Maximum Parsimony Methods.
Molecular Biology and Evolution, 28, 2731-2739.
Tsai.
Lin.
Lee.
Chang.
Linacre, , & Goodwin.
Sequence polymorphism of mitochondrial D-loop DNA in the Taiwanese Han Population.
Forensic Science International, 119, 239-247.
Tuladhar.
Rashid.
Panneerchelvam.
& Nor.
Sequence Polymorphism of Mitochondrial DNA Hypervariable Regions I and II in Malay Population of Malaysia.
Scientific World, 12.
, 24-29.
https://doi.
org/10.
3126_sw.
Wilson.
Allard.
Monson.
Miller, , & Budowle.
Further Discussion of the Consistent Treatment of Length Variants in the Human Mitochondrial DNA Control Region.
Forensic Science Communications, 4.
Wilson.
Allard.
Monson.
Miller, , & Budowle.
Recommendations