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Polymorphism in Non-coding Region of Human Mitochondrial DNA in Persian

Diabetes Type II Patients
*

DorraG1,** Houshmand1M, Shafa Shariat Panahi1M, Larijani2B, Arman1A, Sanati1M H.

Department of Medical Genetics, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran
P.O.Box 14155-6343, Tehran, Iran.Tel: +98 21 4580 390, Fax: +98 21 4580 399, Email:dorraj_gn@yahoo.com
Endocrinology and Metabolism Research Centre, Tehran University of Medical Sciences, Tehran, Iran

Abstract
Population genetic inference would benefit from a better understanding of the variation in the mtDNA coding region,
but, thus far, complete mtDNA sequences have been rare. We determined the nucleotide sequence in the none-coding
region of mtDNA from Persian patients through direct sequencing of the Displacement(D) loop.
The D-loop region is a hot spot for mtDNA alterations and it contains two hypervariable regions. The sequence in the
first Hyper Variable Segment (HVS-I) of the control region has been used as a source of evolutionary information in
most phylogenetic analyses of mtDNA. In order to find for any maternally inherited genetic background of diabetes
type II, the polymorphic sites and also to screen potential variations in the D-loop, the complete non-coding region of
mitochondrial DNA from 7 Azari diabetic patients (clinically diagnosed) and 23 normal controls (people with no
history or family record of diabetes) of the same origin were sequenced.
The sequences were aligned upon the Cambridge Reference Sequence (CRS) and any incompatibilities were recorded
as single base substitution (SBS), numerical changes in Homo-Polymeric C Tract (PCT), insertions or deletions. PCT
changes were present in 52% of normal controls compared to 71% diabetic patients. We found that polymorphism
C150T existed in 42.8% of diabetic patients versus 28.6% in normal controls. Also variation in T16189C was found
in 14.2% of diabetic patients compared to 28.6% in normal samples. mtDNA mutations within the D-loop control
region is frequently reported recurrence in diabetes type II and may be an indicator of mtDNA instability.
Key words: Diabetes Type II, mtDNA, D-loop

Introduction
The human mitochondrial genome is rather small (16.5 kb) and encodes 13 respiratory chain
subunits, 22 transfer RNAs (tRNAs), and two ribosomal RNAs (rRNAs). Mitochondrial DNA is
present at extremely high levels ( 10 2 - 10 5 copies per cell), and the vast majority of these copies
are identical (homoplasmic) at birth (Lightowlers,RN). Expression of the entire complement of
mt genes is required to maintain proper function of the organelle, suggesting that even slight
alterations in DNA sequences could have profound effects (MITOMAP). It is generally accepted
that mtDNA mutations are generated during oxidative phosphorylation through pathways
involving reactive oxygen species (ROS).
The mutation rate for mtDNA is ~10 times higher than that of nuclear genomic DNA
(Wallace,D). The Displacment loop(D-loop), which is 1124 bp in size(Positions 16024-576),is
located between the tRNA genes for proline (tRNAPro) and phenylalanine (tRNAPhe) and is a
non-coding region, and acts as a promotor for both the heavy and light strands of the mtDNA,
and contains essential transcription and replication elements. Despite its functional importance,
this region is believed to be the most rapidly evolving part of the molecule (Upholt,W.B). The
D-loop region is a hot spot for mtDNA alterations, and it contains two hyper variable regions
(HVS-I at positions 16024-16383 and HVS-II at positions 57-372) (Anderson,A). Nucleotide
substitutions accumulate in the mitochondrial genome with a rate considerably higher than for
single- copy nuclear DNA (Brown,M.D). This is most probably due to the lower efficiency of
DNA repair as well as to a higher frequency of DNA replication errors in mitochondrial DNA
(Wilson,MR). Consequently, mtDNA and in particular the non-coding region, is highly
polymorphic. The alterations consist of two major categories: (a) numerical changes in

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Homopolymeric C Tract (PCT), (b) Single Base Substitutions (SBS), Insertions and Deletions.
Within the hyper variable region II a region of micro-satellite-like sequence can be found
(position 208-315). These short tandem repeats, particularly a C-mono-nucleotide track
interrupted by a single thymidine at position 310,has been shown to exhibit length
polymorphism among individuals, as well as variation within an individual, which accompany
the process of aging (Michikaway,Y)Large-scale screening of the mtDNA main control region
in leukocytes from subjects of an Italian population revealed a homoplasmic C150T transition
near an origin of heavy mtDNA-strand synthesis in17% of 52 subjects 99-106 years old, but, in
contrast, in only 3.4% of 117 younger individuals. The authors suggest that the C150T mutation
alters the location of H-strand replication origin and this imparts a replicative advantage to the
mtDNA. But why might C150T be advantageous? One possibility is that it enhances the
immune system in some way, possibly by slowing the turnover of memory T-cells.
Alternatively, because most of the centenarians with the C150T mutation appear to have
inherited it through the germ line, the C150T mutation might simply have been linked to other
variants in the mtDNA that promote longevity (Zhang,J).
Recently, several reports showed a relationship between diseases and mitochondrial
haplogroups (Makino,M.Abe,S). Hofman concluded that certain European mtDNA haplogroups
determine a genetic susceptibility to various disorders. Poulton described a common
mitochondrial variant (16189) that is associated with raised fasting insulin concentration in
several populations.
Type II diabetes is a multifactorial disorder in which variants in mitotochondrial DNA
(mtDNA) could play a role. While diabetes is a major feature in certain mtDNA disease
pathogenic mt DNA mutation have only been identified in a tiny minority of diabetics.
(Poulton,J).
To determine associated alterations in HVS-I and HVS-II with type2 diabetes, the nucleotide
sequence in the D-loop region was determined in Iranian Azari diabetic patients and case
contorol from the same origin.
Materials and Methods:
To address the question whether there are any differences between diabetes typeII patients and
normal controls regarding their D-loop sequence, we sequenced both hyper variable D-Loop
regions from 7 diabetic patients and 23 individuals from the Azari ethnic group. The patients
also belonged to this ethnic group. The presence of diabetes type II was based on clinical
criteria: Onset of diabetes after the age of 30 with no insulin treatment in the first year after
diagnosis. Absence of diabetes in the control was shown by a medical record search. Peripheral
blood samples were obtained and DNA was extracted according to standard techniques. All of
the patients were informed on the aims of the study and gave their informed consents to the
genetic analysis. Peripheral blood samples were obtained and DNA was isolated after lyses of
white blood cells using some DNA extraction kit.
PCR amplified two sequences of 780 bp and 1366 bp length respectively , encompassing the
two hyper variable segments of the mtDNA D-loop to fetch the 359 bp sequence (16024-16383
nt) and 315 bp sequence (57-372 nt) for HVS I and HVS II The nucleotide sequence of the
amplicon was directly determined by automated sequencing 3700 ABI machineThe obtained
mtDNA sequences were aligned in the multiple sequence alignment interface CLUSTALX,
against the Cambridge Reference Sequence.
Results & Discussion
Sequence comparison with the CRS showed C to T transition at position np150 in 42.8% of

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patients and 28.6% of normal controls. Also one T to C transition at position np16189 was
found in 14.2% of patients comparing to 28.6% of the normal samples. Patients with this
mutation had both paternal and maternal family history of diabetes together with insulin
resistance. PCT changes were found in 52% of the control compared to 71% in diabetic patients.
Our comparison of poly-cytidine sequence in diabetic patients with published Cambridge
Sequence revealed that mutations were exclusively found in this region (position 303-315 of the
Cambridge notation, interrupted by a single T). We found that polymorphism C150T existed in
42.8% diabetic patients compared to normal cases (28.6%). This variant was found in 14.2% of
patient with a maternal family history. our data imply that constitutive hyper variable areas such
as the D-loop region represent somatic mutational hot spots. Recently, a polymorphism in the
TAS region at np 16189 has been reported to correlate with an increased risk for type II diabetes
and the 16198 variant lies close to control sequences within the large non-coding region
(Poulton,J). Previous investigators have demonstrated that proteins bind close to this region
(Roberti,M). The presence of the 16189 variant may alter DNA bending hence could influence
interactions between bound proteins controlling either mitochondrial DNA replication or
transcription. Preliminary data suggests that the 16189 variant results in a modest reduction in
mtDNA copy compared with nuclear DNA, the risk associated with this variant was affected by
having a paternal history of diabetes. This would suggest that nuclear genic factor (s) inherited
from the father increased the background risk of the maternally inherited mitochondrial variant
considerably (Poulton,J). Our results showed that 28.6% of normal cases had a T to C transition
in np16189 compared to 14.2% patients with both parental family history of diabetes, Insulin
resistant. Published data show that this polymorphism is as a susceptibility factor in diabetes
typeII and has an association with this disease. Previously, some investigators reported a C150T
mutation in same group of centenarians that there was enrichment for the European haplogroup
J (Debsnedictis,G). Since their report haplogroup J has been associated with longevity in three
other European studies. (Debsnedictis,G. Niemi,AK.). Furthermore, a subset of haplogroup M
mtDNAs has also been associated with longevity in a Japanese study (Tanaka,M). We found
C150T polymorphism in 42.8% of our diabetic patients compared to normal cases (28.6%). Our
patients were 50-60 years old in average and we do not know whether this polymorphism can
promote longevity, but it should be noticed that both mtDNA variations and genetic background
should be taken into account in any consideration of this regard. Replication of mtDNA begins
with the synthesis of the heavy strand (H-strand) using primer RNA, and the 3' termini of primer
RNA were mapped to CSBs I, II and III (Chang,D.D:1985,1987). We found frequent deletions
or insertions only in the CSB II region in71% diabetes patients and 52% of normal controls but
not in other CSBs, suggesting some functional relevance. It is reported that the RNA-DNA
hybrid at CSB II was more stable than those at the other CSBs in vivo (Kang,D).
Acknowledgments
This work was supported by projects 183 and 197 from National Institute for Genetic
Engineering and Biotechnology (NIGEB) and Metabolism Research Centre of Tehran
University of Medical Sciences, Ministry of Science, Research and Technology. We also thank
Molecular Medicine Network for invaluable help.
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