Dr. Mohammadreza Mahdavi
PhD Candidate of Medical Genetics
Figure 11.1 Sites of 'biochemical block' in phenylketonuria, alkaptonuria, congenital hypothyroidism, and oculocutaneous albinism.
Downloaded from: StudentConsult (on 24 May 2011 06:54 AM)
© 2005 Elsevier
Figure 11.2 Facies of a male with phenylketonuria; note the fair complexion.
Downloaded from: StudentConsult (on 24 May 2011 06:54 AM)
© 2005 Elsevier
PKU
y Autosomal recessive genetic disorders
y Frequency : 1 in 10000 and in Caucasians live births.
y Severe mental retardation
y Adopting low-phenylalanine dietary treatment is main methods in preventing
mental retardation.
PAH Gene
y PAH gene is located on q22–q24.1 regions of chromosome 12.
y 13 exons and 12 introns
y 90 kb long.
l
y More than 500 different mutations
Aim of the Study
y The aim of this study was to identify the
frequency of five common mutations on PAH
gene among patients with PKU in Mazandaran
and Golestan provinces.
Samples
y A total of 40 unrelated PKU patients were identified
y 22 had Mazandarani origin
y 18 had Golestani origin (including Turkmens)
Methods
y PCR
PCR- RFLP method was used to detect five common
y
y
y
y
y
mutations including:
IVS10-11G>A (Dde I)
R261Q (Hinf I)
R252W (Ava I)
R261X( Dde I)
IVS11+1G>C (Dde I)
DNA Isolation
Genomic DNA was extracted from leukocytes
using Qiagen DNA extraction kit
Amplification of Intron 10 for the
IVS10 11G>A Mutation
detection of IVS10-11G>A
.‫ ﺟﻔﺖ ﺑﺎزي از اﺳﺘﻔﺎده ﺷﺪ‬444 ‫ در اﺑﺘﺪا ﺟﻬﺖ ﻳﻚ ﻗﻄﻌﻪ‬PCR ‫ از‬9
.‫ در دﺳﺘﮕﺎه ﺗﺮﻣﻮﺳﺎﻳﻜﻠﺮ اﻧﺠﺎم ﭘﺬﻳﺮﻓﺖ‬25µ ‫ ﺑﺎ ﺣﺠﻢ ﻧﻬﺎﻳﻲ‬9
MATHERIAL
VOLUME
ddH2O
16.57 μl
Buffer
ff PCR
C (10X)
2 5μll
2.5
MgCl2(50mM)
0.7 μl
dNTPs(10mM)
0.5 μl
Primer Forward (10μM)
(5-TAGACATTGGAGTCCACTCTC-3 )
Primer Reverse (10μM)
(5-AATCGGGGTGAGATGAGAG-3)
T Polymerase
Taq
P l
(5U
(5Unit/
it/μ))
1.0 μl
DNA
2. 0μl
1.0 μl
0 2 μll
0.2
PCR Program
Time
Temperature
Number of cycles
5′
94
Initial Denaturation
45″
94
Denaturation
45″
60
Annealing
45″
72
Extension
5′
72
ّ◌Final Extension
1
35
1
Dde I enzyme was used for the detection of
IVS 10-11 G>A Mutation
For the identification of IVS 10-11 G> A mutation Dde I
restriction enzyme was used
MATERIALS
VOLUME
ddH2O
6.0 μl
Buffer (10X)
1.5 μl
Dde I
0.5 μl
PCR Product
7 μl
l
l
h
Gel Agarose
Electrophorasis
PCR-RFLP Product were analysed on 2%
Agarose gel .
Result of a PCR‐RFLP reaction for the IVS
detection of 10 11 G>A Mutation
detection of 10-11
1 2 3 4 5 6
338bp
246 and 92 b (is not bp
(i t visible in this picture)
Lane 1: 100bp Ladder
Ladder, Lane 2 and 3 patients
heterozygote for IVS 10-11 G> A mutation, Lane 5
Normal subject, Lane 6 control negative.
Allelic frequencies of five investigated mutations among patients
Mutation
Exon/Intron
Number of alleles
Frequency %
IVS10-11G>A
I 10
22
27.5
R261Q
E7
3
3.75
R261X
E7
1
1.25
R252W
E7
0
0
IVS11+1G>C
I 11
0
0
26
32.5
Numbers of identified
mutations
The genotype of PKU patients in Mazandaran
and Golestan provinces
Genotype
Number of Mazandarani
Number of Golestani
patients
patients
3
6
9
2
2
4
R261Q/ R261Q
1
0
1
R261Q/U
1
0
1
R261X/ U
1
0
1
U/U
14
10
24
IVS10 11G>A/ IVS10
IVS10-11G>A/
IVS10-11G>A
11G>A
IVS10-11G>A/ U
Total
Discussion
y In subjects with Golestani Origin only IVS10-11G>A
mutation was found.
y In Mazandran province three different mutations
were identified (IVS10-11G>A,
(IVS10 11G>A R261Q,
R261Q and R261X)
The most frequent mutations in Iran
q
Province Name
Number of
investigated
Cases
The most
frequent
mutation
Frequency (%)
Khuzestan
40
IVS10-11G>A
10
Azerbaijan
40
IVS10-11G>A
23.75
Azerbaijan
44
IVS10-11G>A
19.3
Zanjan and
Ghazvin
39
p.P281L
p
P281L and
R176X
10 25
10.25
Isfahan
34
R252W
15.38
Mazandaran
22
IVS10-11G>A
18.2
Golestan
18
IVS10-11G>A
38.8
Conclusion
y IVS10-11G>A mutation is the most frequent mutation
in most of the provinces in Iran.
y The highest frequency of IVS10-11G>A mutation in
Iranian PKU patients was reported in the present
study and in Golestan Province (38.8 ).
The most frequent mutations in Middle East
The most frequent mutations in Middle East
Province Name
Number of
investigated Cases
The most frequent
mutation
Frequency (%)
Israeel
78
IVS10
11G A
IVS10-11G>A
18.8
Turkey
44
IVS10-11G>A
IVS10
11G A
32
Israeel
18
A403V
23.3
Mazandaran
22
IVS10-11G>A
18.2
Golestan
18
IVS10-11G>A
38.8
(Non-Ashkenazi Jews)
(Ashkenazi Jews)
Conclusion
y IVS10-11G>A mutation is the most frequent mutation
in the middle east.
y The highest frequency of IVS10-11G>A mutation in
PKU patients in the middle east was reported in the
present study and in Golestan Province (38.8 ).
11G>A
High frequency of IVS10
IVS10-11G>A
mutation
y Genetic drift
y Gene flow from other populations (Turkish Azeri
population)
R
d i
Recommendation
different mutations are responsible for PKU disease in
north of Iran.
further studies (Sequencing) are recommended to
identify all of the mutations on PAH gene in the
region.
region