Investigation of the Mitochondrial Haplogroups in A Selective Population of Isfahan Province- Juniper Publishers
Juniper Publishers- Journal of cell Science
Abstract
In order to investigate the mitochondrial haplogroups
in a selective population of Isfahan province, 96 unrelated men in
Isfahan province with at least two generations in this province were
token blood. Then DNA was extracted and quality assessed. 25 coding SNPs
defining the major haplo groups that occur in Africa, Western Eurasia
and Eastern Eurasia were selected and combined into two multiplex
genotyping assays. Each one consisting of a PCR step and a SNaPShot
step. Then for support of our detective haplo groups by SNaPShot system,
D-Loop region was sequenced in some samples. : In this province Western
Eurasian haplo groups were predominant. Haplo group U and H (22%, 21%)
and then T, J and U8b (11%, 10% and 7%) had the most frequency. Eastern
Eurasian haplo groups (C, D, R9, R11, M and N) were present at a lower
frequency and a North African haplo group (M1) at frequency of 1%.
Keywords: mtDNA; SNP; Haplo group; Isfahan provinceIntroduction
Human mitochondrion has a DNA independent of nucleus
DNA called mtDNA. mtDNA is a dabble strand circular molecular that has
some special feathers that make it useful in human evolution studies. It
has maternal inheritance [1], higher rate of mutation than nuclear DNA [2], high copy numbers and lack or recombination[3].
Due to these special feathers, mtDNA are widely used as a useful tool
in studies like population history, medical genetics, genetic genealogy,
and genetic forensic. Analyzing mtDNA patterns in modern human use for
tracing genetics journey of early women. By mtDNA studies researchers
can trace a targeted maternal ancestry along time and defining maternal
ancestor in a selective population.
A haplogroup is a genetically group of population
that has common ancestors in maternal/paternal lines. Mutated
nucleotides in mitochondrial genome are transferred as polymorphisms
from mother to boy and girl children. The whole polymorphisms in an
individual mtDNA is called mitochondrial haplotype. Varies haplo types
in different ancestries create branches of mtDNA phylogenic tree and
consequently aggregation of haplo types in this tree creates clusters as
haplo group [4].
mtDNA phylogenic tree is classified in four macro haplo groups named L,
M, N and R. each one has some haplo groups and each haplo group has
some sub haplo groups.
mtDNA and Y chromosome haplo groups studies are
useful in cases like investigation on human colonization patterns from
different continents and understanding origin and genetic structure of
different populations.
The Purpose of this study is defining of each haplo
group frequency in a selective population of Isfahan province, a central
province in Iran.
Material and Methods
96 unrelated men inhabited in Isfahan province with
at least two generations in this province were token blood, based on the
number of population of each city. Then DNA was extracted by RGDE
method [5] and quality assessed by nano photometer and electrophoresis. We selected 25 coding SNPs [6] defining 32 of the major haplo groups that occur in Africa, Western Eurasia and Eastern Eurasia (Figure 1). SNPs combined into two multiplex genotyping assays. Each one consisting of a PCR step and a SNaPShot step.
Multiplex PCRs were performed in a reaction volume of
6μl, 0.35units of Taq DNA polymerase, 1.2μl watery genomic DNA in which
contained 1X Gene Amp PCR buffer, 100μM of each dNTPs 5mg/ml and 15
pairs PCR primers for multiplex! (Table1) and 12 pairs PCR primers for multiplex 2 (Table 2).
The reactions were performed in a ABI Bio system applied 2730 with
conditions: an early denaturation at 95 °C for 10minuet and 30 cycles of
94 °C for 15 seconds, 60 °C for 45seconds and a final extension at 60
°C for 5minuet. Then PCR products were purified by adding 1.5μl ExoSAP
to them and incubation at 37 °C for15 minuet and 80 °C for 15 minute.
Multiplex SNaPShot were performed in a reaction volume of 5μl, which
contained 1μl Ready Reaction Mix, 1μl purified PCR product and
extensions primers (Tablel 3& 2).
The reactions were performed in conditions: 96 °C for 2minutes and 25
cycles of 96 °C for 10 seconds, 50 °C for 5seconds and 60 °C for 30
seconds. The reaction products were purified by adding 1μl CIP to them
and incubation at 37 °C for 45 minutes and 75 °C for 15 minutes.
prepared a mixture of l^l purified extension product, 8.8 μl Hi- Di for
maimed and 0.2 μl GeneScan-120 LIZ size standard and ran samples in
genetic analyzer ABI 1330XL with POP-7 polymer. Results were analyzed
using Gene Mapper ID version 3.2 software. The results of SNP patterns
in two assays was determining mitochondrial haplo groups in samples of
the study. Then for support of our detective haplo groups by SNaPShot
system, D-Loop region was sequenced in some samples by genetic analyzer
ABI 1330XL.
Results
96 mtDNA samples from Isfahan province were analyzed and 18 haplogroups were found (Figure 2).
94% of this population belonged to Western Eurasian haplo groups and
only 5% belonged to Eastern Eurasian haplo groups (C, D, R9, R11, M and
N). So Western Eurasian haplo groups were predominant. Haplo group U and
H (22%, 21%) and then T, J and U8b (11%, 10% and 7%) had the most
frequency and Eastern Eurasian haplo groups and a North African haplo
group (M1) were present at a lower frequency.
Discussion
Previous studies indicated a high frequency of 77% of
Asian mtDNA is belonged to haplogroup M. Since Iran is located in Asia
it is expected that this haplo group has a high frequency in Iran. But
results of Houshmand et al. 2004 study showed a low frequency of this
haplo group (0-4%). This study found the same results and only three
cases of persons who take part in this study had haplo group M.
In other side about 99% of mtDNA in European
population belong to at least one of the nine haplogroups: H, U, J, T,
K,I, V, W, and X. Six of them (H, J, T, K, I and w) are especial for
European population and probably originated from Caucasoid and
genetically separated from ancestors of African and Asian [7].
Studies about Iran country showed a high frequency of Haplo group U,
also in this study had the most frequency (22%). This haplo group is
much older than the others and estimated to originate about
51,000-67,000 years ago [7].
It seems that this haplo group originated from Africa and then expanded
into Middle East and Europe. In the other hand haplo groups H, T, J and
K had the most frequency after haplo group U in Isfahan province. It is
estimated that originated about 8,000-30,000 years ago [7,8].
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