Download the frequency of the factor v leiden (f5)1691g>a allele among sri

THE FREQUENCY OF THE FACTOR V LEIDEN (F5)1691G>A ALLELE AMONG
SRI LANKAN PATIENTS WITH THROMBOEMBOLISM
........*........
DESIGN AND IMPLEMENTATION OF A NOVEL GENOTYPING ASSAY FOR
CYP2C9, CYP4F2 AND GGCX POLYMORPHISMS TO PREDICT WARFARIN
MAINTENANCE DOSE
DESIGN AND IMPLEMENTATION OF A TETRA PRIMER AMPLIFICATION
REFRACTORY MUTATION SYSTEM (T-ARMS) POLYMERASE CHAIN
REACITON GENOTYPING ASSAY FOR CYP2C19*2 AND CYP2C19*17
POLYMORPHISMS
......*.......
MOLECULAR CYTOGENETIC CHARACTERIZATION OF THE FIRST
REPORTED SRI LANKAN CHILD WITH A DE NOVO 9p INVERTED
DUPLICATION (p13.3;p23)
BY
IMAYA UVINI HEWA GODAPITIYA, B.Sc
FMC/GD/02/2012/03
DISSERTATION SUBMITTED TO
THE UNIVERSITY OF COLOMBO, SRI LANKA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS OF THE
MASTER OF SCIENCE IN GENETIC DIAGNOSTICS
AUGUST 2014
CERTIFICATION
I certify that the contents of this dissertation are my own work and that I have acknowledged
the sources where relevant.
…………………………………………
Signature of the candidate
This is to certify that the contents of this dissertation were supervised by the following
supervisors:
MUTATION REPORT
…………………………….
…………………………….…
Dr. U.N.D. Sirisena
Prof. V.H.W. Dissanayake
PHARMACOGENOMICS REPORT
…………………………….
…………………………….…
Dr. K.T. Wettasinghe
Prof. V.H.W. Dissanayake
MOLECULAR CYTOGENETICS REPORT
…………………………….
Ms. I. Kariyawasam
…………………………….
Prof. V.H.W. Dissanayake
…………………………….…
Ms. V. Udalamaththa
ACKNOWLEDGEMENTS
In the year 2010, I was a trainee at Asiri Centre for Genomics and Regenerative Medicine
(ACGRM) working under Professor Vajira H. W. Dissanayake when he told me about this
master’s program. As I did not have any working experience, I prepared myself for two years
so that I would be well qualified for this program and got enrolled for the second intake in
2012. This project was supported by the NOMA grant funded by NORAD in collaboration
with the University of Colombo (UoC), Sri Lanka and the University of Oslo (UiO), Norway.
The work presented in this dissertation was carried out at the Human Genetics Unit, Faculty
of Medicine, University of Colombo. I would like to express my sincere gratitude to
Professor Vajira H. W. Dissanayake, my senior supervisor, Professor in Anatomy and
Medical Geneticist at Asiri Centre for Genomic and Regenerative Medicine (ACGRM) and
Human Genetics Unit (HGU), Faculty of Medicine for encouraging me and motivating me
for the best and preparing me for the worst and providing access for all the available training
programs, conferences to help us learn more than what the book says. The comments,
suggestions and advice received from you are greatly appreciated.
The constant guidance received from Professor Rohan Jayasekera (Director, Human Genetics
Unit (HGU), Faculty of Medicine, UOC) during the master’s program was extremely
valuable for personal and career development.
I would like to show appreciation and gratitude to my supervisors, Dr. U. N. D. Sirisena, Dr.
K. T. Wettasinghe, Ms. I. Kariyawasam and Ms. V. Udalamaththa for their constant support
and guidance in laboratory work as well as in reviewing my manuscripts. Appreciations goes
to Miss. P. K. D. S. Nisansala for her active support in updating the database and keeping
every patient record in order and to Mr. Sisira Perera, Mr. Nihal De Saram and Mrs.
Sakunthala Bandaranayake for their patience in letting me stay in the laboratory till late hours
so I could finish my PCR procedures. My thanks go out to Mr. Nazeel Mohomed for
providing the necessary assistance in computer software and hardware.
The clinical evaluation of the patients would not have been possible without Dr. Niluka
Dissanayake. Your input and contributions from the beginning of the research and helping me
out with the clinical aspects of this project and for being a genuine colleague and a close
friend is greatly appreciated.
I deeply appreciate the constant support and guidance given by my fellow MSc friends
Shayini, Damitha and Channa throughout the MSc course.
My immense gratitude and love to my super parents for their love, sacrifice, support,
encouragement and timely advice throughout my life. You always believed in me and showed
me how to achieve the impossible. You both are my inspiration and my motivation. My Love
and gratitude goes out to my big brother for providing me the luxury to work without stress
and giving me timely advice and encouragement to live life to the fullest and also to my sister
for the love, advice and support.
Lastly I would like to thank Professor Eirik Frengen, Associate Professor, UiO/OUH and
other faculty members of the UOC and Department of Medical Genetics, OUH/UiO Norway
for giving me hands-on experiences in various techniques and softwares and everyone at the
Human Genetics Unit and Anatomy department, Faculty of Medicine who have helped me in
various ways throughout the duration of my MSc and all my loving friends for making these
two years memorable and enjoyable.
MUTATION REPORT
THE FREQUENCY OF THE FACTOR V
LEIDEN (F5)1691G>A ALLELE AMONG
SRI LANKAN PATIENTS WITH
THROMBOEMBOLISM
ABSTRACT
Background: Thrombophilia, a condition which predisposes to arterial and/or venous
thrombosis occurs due to either inherited or acquired risk factors. Resistance to activated
protein C (APC) caused by Factor V Leiden (F5) 1691G>A (rs6025) variant is an inherited
risk factor commonly associated with venous/arterial thrombosis and adverse pregnancy
outcomes. The objective of our study was to determine the frequency of the Factor V 1691A
allele among Sri Lankan patients with Thromboembolism.
Methods: The F5 1691G>A test results of 887 patients with various thrombotic events
referred for thrombophilia screening to the Human Genetics Unit, Faculty of Medicine,
University of Colombo from January 2007 to December 2013 were retrospectively analysed.
Results: The frequency of the F5 1691G>A variant allele among the Sri Lankan
thrombophilic patients was 1.3%. Further classification based on the indication for referral
showed that the F5 1691G>A variant allele was highest among patients with venous
thrombotic events (3.6%), followed by arterial thrombosis (2.6%) and pregnancy
complications (0.7%).
Conclusion: Compared to other studies from the Indian sub-continent, the frequency of the
F5 1691G>A variant allele was relatively lower among the Sri Lankan thrombophilic patients
with thromboembolism. Screening for F5 1691G>A variant should be undertaken in the
diagnostic work up of patients referred for various thromboembolic events. Screening for
asymptomatic family members should be offered in cases where the variant alleles are
detected so that proper counselling and management can be provided to prevent future
thromboembolic events.
KEYWORDS: Thrombophilia, Thromboembolic disorders, Factor V Leiden, FVL, F5
1691G>A, variant
1
INTRODUCTION
Thrombophilia is a multifactorial disorder which predisposes to arterial and venous
thrombosis. It can occur due to either inherited or acquired risk factors. Factor V Leiden
(FVL) (F5) 1691G>A (rs6025) variant, is the most common genetic variant associated with
inherited thrombophilia, activated protein C (APC) resistance and venous thromboembolism
(VTE). Factor V Leiden thrombophilia (FVL) is inherited as an autosomal dominant
condition.
Factor V is one of the proteins in the coagulation pathway. The variant is caused by a
substitution of nucleotide G (Guanine) by A (Adenine) at position 1691 (1691G>A) in the F5
gene which is located on the first chromosome at 1q23 locus and is composed of 81,578 bps.
Factor V levels are controlled by thrombin-dependent activation, and APC-dependent
inactivation. Along with thirteen coagulation factors, activated Factor V together with
activated factor X and Ca2+ ion form the prothrombinase complex, which promotes the
conversion of prothrombin or Factor II to thrombin following which thrombin convert’s
fibrinogen to fibrin. Protein C is an anticoagulant that cleaves two activated coagulant
factors, Factor VIIIa and Va, there by inhibiting the conversion of factor X to Xa and
Prothrombin to thrombin to inhibit excessive clotting. The mutation in the Factor V molecule
renders Factor Va resistance to proteolysis by activated protein C thus the term activated
protein C resistance leads to a thromboembolic state (1, 2).
People with Factor V Leiden variant are at risk of developing venous thromboembolic
disorders (VTE) including deep vein thrombosis (DVT) and pulmonary embolism (PE). VTE
is a significant cause of morbidity and mortality in many countries with an annual incidence
of 1/1000. It is very rare for FVL thromboembolism condition to cause the formation of clots
in arteries that can lead to stroke or heart attack, though transient ischemic attack is common.
This disease displays incomplete dominance; those who are homozygous for the mutated
2
allele are at a higher risk for FVL thrombophilia than those that are heterozygous for the
mutation (3). Factor V Leiden variant is known to increase the risk of venous
thromboembolisms but by itself does not appear to increase the risk of arterial thrombosis (4).
Women with Factor V Leiden have a considerably increased risk of thrombosis in pregnancy
in the form of deep vein thrombosis and pulmonary embolism. They also may have a small
increased risk of preeclampsia, having low birth weight babies, miscarriage and stillbirth due
to either clotting in the placenta, umbilical cord, or the fetus (5, 6). Acquired risk factors
comprise lupus anticoagulants, pregnancy, use of contraceptives, major surgeries, cancer,
inflammations, and etc.(7).
Published reports indicate high frequency of F5 1691G>A variant among venous
thromboembolic patients and healthy controls in Caucasian (45%) and Mediterranean
populations (50%) (7) but relatively lower frequency among African and Asian populations
(7-11). It is reported that the frequency of FVL in Indian patients with venous
thromboembolism was around 3% (9). Previously published Sri Lankan studies have reported
the frequency of the variant allele of F5 1691G>A variant with reference to the country’s
major ethnic groups: 2.0% (Sinhalese) 3.0% (Tamils) and 2.0% (Moors) (12).
Genetic risk factor assessment has become an essential component of the diagnostic
evaluation of patients who present the signs and symptoms of thromboembolism. The
molecular detection of this variant can be useful clinically in the identification of
thrombophilic diseases (13).
The objective of our study was to determine the frequency of the Factor V 1691A allele
among Sri Lankan patients with Thromboembolism.
3
MATERIALS AND METHODS
Subjects
We retrospectively analysed the clinical and genotype data of the patients with various
thrombotic events referred for thrombophilia screening manifesting venous, arterial
thromboembolism and pregnancy complications to the Human Genetics Unit, Faculty of
Medicine, University of Colombo, Sri Lanka from the period of January 2007 to December
2013. Written informed consent was obtained from all the patients prior to genetic testing.
Genotyping Assay
Genomic DNA, isolated from the venous blood, were collected into EDTA containing tubes
and stored at –20°C prior to DNA extraction. DNA extraction was done using QIAamp®
DNA mini kits (Qiagen Ltd., UK) according to the manufacturer’s protocol. The mutation
was detected using Multiplex Polymerase Chain Reaction followed by Restriction Fragment
Length Polymorphism (PCR/RFLP) assay using Mnl1 endonuclease digestion as previously
described (13).
RESULTS
The total number of patients tested from January 2007 to December 2013 at the HGU was
887. Of them 519 (58.5%) were female and 368 (41.5%) were male. The distribution of
patients according to the indication for referral is shown in Table 1.
The overall frequency of F5 1691G>A variant in the study population was 2.6% (23/887) and
frequency of the variant allele (A) was 1.3%. Further classification according to the
indication for referral showed a frequency of 3.6% (venous thromboembolism), 2.6% (arterial
thrombosis), and 0.7% (pregnancy complications). The mean age of all the patients referred
for thrombophilia was 30 years (age range 1- 73 years). The racial distribution of the patients
referred included: 769 Sinhalese, 61 Tamils, 56 Moors and 1 Burgher.
4
The summarized genotype frequencies of the F5 1691G>A variants in Sri Lankan
thrombophilic patients are shown in Table 2. Symptomatic family members of those who
harboured the F5 1691G>A variant were tested. The clinical details of the patients with F5
1691G>A variant and the results of asymptomatic family members who were tested are
summarized in Table 3.
Table.1 Distribution of patients according to the indication for referral
Thrombotic event
Cerebro Vascular Accident (CVA)
Myocardial Infarction
Arterial
Frequency
311
38
Young Stroke
32
Other arterial thrombosis (i.e. Left ophthalmic artery
obstruction, L/Superficial femoral artery obstruction,
L/Carotid artery thrombosis)
07
Deep Vein Thrombosis (DVT)
Pulmonary Embolism (PE)
DVT+PE
Cerebro Venous Thrombosis
Abdominal Vein Thrombosis
Other (i.e. neck/eye vein thrombosis, upper limb veins...)
166
31
16
27
65
30
Total
388 (43.7 %)
VTE
335 (37.8 %)
Pregnancy
Complications
Recurrent Pregnancy Loss(1st trimester,2nd trimester from
p2c0-p7c0
Bad Obstetric History
Sub-fertility
IUD/Failed IVF
Pre-eclampsia and Recurrent pregnancy loss (p2c0)
143
3
3
2
2
154 (17.4 %)
Other
B/L Avascular necrosis of the femoral heads / Hip
Recurrent thrombosis of AVF sites / hemorrhage
Sudden loss of vision - L/eye
Central retinal occlusion
Superficial thrombophlebitis
2
3
1
3
1
10 (1.1%)
887 (100 %)
5
Table 2. Genotype frequencies of the F5 1691G>A variant in Sri Lankan thrombophilic
patients
Genotype frequency [%]
SNP
Gene
Genotype
(SNP ID)
F5
Venous
Thrombosis
Arterial
Thrombosis
Pregnancy
complications
1691G>A
GG
323 (96.43%)
378 (97.4%)
153 (99.3%)
(rs6025)
GA
12 (3.6%)
10 (2.6%)
1 (0.7%)
AA
0 (0.0%)
0 (0.0%)
0 (0.0%)
335 (100%)
388 (100%)
154 (100%)
Total
DISCUSSION
According to our data it was observed that the genotype frequency (GA) was 2.6% and
frequency of the variant allele (A) was 1.3% which is lower when compared to reported
frequency in south Indian populations (3%) (9).
Recognized thrombophilia manifestations are mainly venous territorial thromboembolism.
Association between FVL and arterial thromboembolism is debatable but not uncommon.
Our F5 1691G>A variant positive patients presented arterial thrombosis (anterior non ST
elevation myocardial infarction, cerebro vascular accidents, Left/middle cerebral artery
infarction, myocardial infarction, Right/parietal lobe infarction, Right/S frontal and basal
ganglion infarction, sensory stroke, young stroke), pregnancy complications (recurrent
miscarriages and pre eclampsia) and venous thromboembolisms
(cerebral venous
thrombosis, deep vein thrombosis, deep vein thrombosis bilateral (DVT-B/L) lower limb, left
side ( L/S) retinal vein thrombosis, recurrent DVT, subarachnoid haemorrhage, thrombosis of
left internal jugular vein , unprovoked DVT, venous sinus thrombosis).
6
Heterozygous
F5 1691G>A variant increases the risk of developing a first deep vein
thrombosis (DVT) by 5 to 7 fold (14, 15). The homozygous state increases the risk by 25 to
50 fold. However there is no evidence that heterozygosity for Factor V Leiden increases the
overall mortality rate (4, 16, 17). If the heterozygous form of F5 1691G>A is present, the
lifetime risk of developing a DVT is approximately 10%, but may be higher when having
close family members who have had DVT (15). In people with homozygous Factor V Leiden
or with combined inherited thrombophilia, the risk of venous thromboembolism is increased
to 20 to 50 times higher although whether if the risk of death is higher is not clear (18, 19).
Previously published data suggests that approximately 1 in every 1000 people will develop
DVT or pulmonary embolism (PE) each year, and this increases from about 1 in 10 000 for
those in their twenties to about 5 in 1000 for those in their seventies. Most people with F5
1691G>A variant have additional risk factors that contribute to the development of
thrombosis such as obesity, cancer, hospitalization, surgery or trauma, pregnancy and taking
oral contraceptives. Having F5 1691G>A alone does not appear to increase the risk of
developing venous thrombosis (15).
According to two reported meta-analyses a small but significant association with arterial
thrombosis also exists. Besides coronary artery disease, an association of FVL with ischemic
colitis is also reported. The relation of FVL with ischemic stroke is controversial, especially
in young adults as there were no significant correlation with previous meta-analysis studies
(20).
Previous studies indicate that pregnancy can be associated with a 5- to 6-fold increase in the
risk of VTE and is the leading cause of morbidity and mortality in pregnancy and the
postpartum period. VTE occurs in approximately 1 in 1500 pregnancies, and up to one fourth
of untreated VTE may PE. Women with a personal history of VTE in an earlier pregnancy
have a high incidence of FVL than those who have never had a VTE. Women who are
7
pregnant and heterozygous for FVL have a 5- to 10-fold increase in the risk of VTE, while
those who are homozygous have a 50- to 100-fold increased risk (21). Other maternal
complications of FVL comprise the hypertensive disorders of pregnancy and placental
abruption. Fetal complications such as miscarriage, intrauterine fetal demise (IUFD),
placental abruption, and intrauterine growth retardation (IUGR) have also been associated
with FVL. Previously published data indicate that female heterozygote’s for F5 1691G>A
variant are at increased risk for severe pre eclampsia and other adverse pregnancy outcomes
as well as DVT (22, 23). But a recent study reported in Sri Lanka in parallel to other studies
(24) shows that there was no statistically significant association of any particular
thrombophilic polymorphism in patients with first-, second- or third trimester pregnancy
losses (25).
A previous report shows that patients with FVL present a thrombophilic tendency that may be
enhanced during an inflammatory episode during sepsis, infection, or inflammatory bowel
disease. Damage to the endothelium, exposure of adhesion molecules, and the involvement of
procoagulant components involved in inflammation with activation of leukocytes and
platelets and FVL play a key role in up regulating the underlying thrombophilic state even in
patients with the heterozygous FVL mutation (26).
There are other complications that could occur or increase the incidence due to FVL.
Heterozygote’s have a 2- to 3-fold increased risk for central venous catheter-related
thrombosis in patients with advanced or metastatic breast cancer and those who are
undergoing allogeneic bone marrow transplantation. It shows that the risk for VTE is
increased more than 100-fold in women homozygous for Factor V Leiden who use oral
contraceptives (27).
8
It has been reported
that FVL-associated phenotypes seems to have had considerable
evolutionary impacts such as protection from acute blood loss, menstrual blood loss,
proneness to pregnancy complications and coronary heart disease (20).
It is reported that in those with FVL, the risk of venous thromboembolism is three to four
times higher if there is a positive family history. The risk of a first event is two to three times
higher in people with a family history of thrombosis in a first-degree relative. The risk is four
times higher when multiple family members are affected, at least one of them before 50 years
of age (28).
In conclusion with regard to all the information available, screening for F5 1691G>A variant
should be undertaken in the diagnostic work up of Sri Lankan patients referred for venous
and arterial thromboembolic events as well as pregnancy related thrombotic complications.
Screening for asymptomatic family members should be offered in cases where the variant
alleles are detected so that proper counselling and management can be provided to prevent
future thromboembolic events.
9
Table 3. Clinical data of patients and asymptomatic family members who were positive for the F5 1691G>A variant
Index
Patient
135
166
262
511
65
830
865
958
959
Indication for Referral
Thrombotic event
Sex
Age
(Years)
Genotype
Relationship
Myocardial Infarction
Asymptomatic
Asymptomatic
Pre symptomatic screening
Pre symptomatic screening
Recurrent DVT
Asymptomatic
Venous sinus thrombosis
Asymptomatic
Asymptomatic
L/S cerebral infarction
Pre symptomatic testing
Pre symptomatic testing
Thrombosis of left internal jugular vein
Asymptomatic
Cerebro Vascular Accident
Pre symptomatic screening
Pre symptomatic screening
Pre symptomatic screening
Subarachnoid haemorrhage
Presymptomatic screening
Pre symptomatic screening
Pre symptomatic screening
Arterial
Male
Female
Female
Male
Male
Male
Male
Female
Male
Female
Male
Male
Female
Female
Female
Male
Female
Male
Male
Female
Male
Female
Female
9
39
6
27
75
45
13
36
76
6
46
54
10
50
20
30
50
16
14
40
3
57
26
GA
AA
GA
AA
GA
GA
GA
GA
GA
GA
GA
GA
GA
GA
GA
GA
GA
GA
GA
GA
GA
GA
GA
Index case
Mother
Sister
Uncle
Grandfather
Index case
Son
Index case
Father
Daughter
Index case
Brother
Daughter
Index case
Daughter
Index case
Mother
Brother
Brother
Index case
Son
Mother of a Index Negative
Sister of a Index Negative
Venous
Venous
Arterial
Venous
Venous
Venous
10
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12
PHARMACOGENOMICS REPORTS
DESIGN AND IMPLEMENTATION OF A
NOVEL GENOTYPING ASSAY FOR
CYP2C9, CYP4F2 AND GGCX
POLYMORPHISMS TO PREDICT
WARFARIN MAINTENANCE DOSE
ABSTRACT
Introduction: Warfarin (brand name Coumadin) is the most widely prescribed oral
anticoagulant drug in the world which is used for a wide range of diseases and conditions.
Warfarin has a constricted therapeutic index and a wide inter-individual unpredictability in
dose requirement and at risk with under dosing or overdosing. It has been observed that
Asians require more warfarin to achieve the same level of anticoagulation than Caucasians
independent of age, sex, and body mass index (BMI). Studies have shown that single
nucleotide polymorphisms in the cytochrome P450 2C9 (CYP2C9), GGCX, CYP4F2 V433M
and vitamin K epoxide reductase (VKOR) genes have a significant effect on warfarin dose
requirement. Although testing for CYP2C9*2 and CYP2C9*3 for warfarin therapy is being
carried out in Sri Lanka, CYP4F2 V433M and GGCX polymorphisms have not been tested.
Objective: The objective of this study was to design and implement a genotyping assay for
CYP2C9*2,*3,*5,*6, CYP4F2 and GGCX to predict warfarin maintenance dose.
Materials and Methods: Clinical samples of pre-purified hgDNA from 20 de-identified
patients were used to implement and optimize a tetra amplification refractory mutation
system (T-ARMS) multiplexed polymerase chain reaction (PCR) method. Two approaches
were taken; single variant T-ARMS PCR and two variant multiplexed PCR. The primers
were designed to have a specific 3’ end to the variants and troubleshooting, optimization and
sequencing were performed to obtain the optimal results.
Results: The desired specific bands were obtained by the single variant T-ARMS PCR
method and except for one variant; two variants multiplexed PCR method also gave the
expected bands.
1
Discussion and Conclusion: The optimum PCR conditions and parameters were obtained by
a series of troubleshooting. Further optimizations are required to achieve substantial optimal
parameters and obtain reproducibility of the two variants multiplexed PCR method, as this
would be useful for routine laboratory practice. As the genetic variants of CYP2C9, CYP4F2
and GGCX genes show a significant association with the interindividual therapeutic dose of
warfarin, it is important to implement this genotyping assay as it would be a medically and
economically beneficial, cost effective pharmacogenomic platform so that the therapeutic
dose can be personalized in patients.
Key words: Warfarin, CYP2C9, CYP4F2, GGCX, T-ARMS multiplexed PCR
2
INTRODUCTION
Warfarin (brand name Coumadin) is the most widely prescribed oral anticoagulant drug in
the world which is used for a wide range of diseases and conditions. It is used for the
prevention and treatment of arterial and venous thrombo-embolic diseases including Deep
Vein Thrombosis (DVT), Pulmonary Embolism (PE), Ischemic Stroke, Myocardial Infarction
(MI), and Arterial Fibrillation. Warfarin has a constricted therapeutic index and a wide interindividual unpredictability in dose requirement and at risk with under dosing or overdosing,
respectively (1, 2). Multiple factors including age, sex, weight, liver function, concomitant
drugs, certain disease states, diet and genes, affect its dose response (3). It has been observed
that Asians required more warfarin to achieve the same level of anticoagulation than
Caucasians and that this is independent of age, sex, and BMI. (4) Studies have shown that
single nucleotide polymorphisms (SNP) in the cytochrome P450 2C9 (CYP2C9), GGCX,
CYP4F2 V433M and vitamin K epoxide reductase (VKOR) genes have a significant effect on
warfarin dose requirement (1).
Cytochrome P450, (CYP2C9), which is located in chromosome 10q region, a major isozyme
of the CYP2C subfamily in human liver, constitutes about 20% of the total human liver
microsome P-450 content and metabolizes ~ 10% of therapeutically important drugs, some
such as warfarin with a narrow therapeutic index (5). This is mostly responsible for the
metabolism (clearance) of S- warfarin (6). Several finding suggests that the human CYP2C9
gene is highly polymorphic (http://www.imm.ki.se/CYPalleles/cyp2c9.htm) (7). CYP2C9
variants such as CYP2C9*2 (rs1799853, 430C>T), CYP2C9*3 (rs1057910, 1075A>C)
variants lead to reduced enzyme activity and more prevalent in Asian and Caucasian
population and CYP2C9*5 (rs28371686, 1080C>G), CYP2C9*6 (rs9332131, 818delA) leads
to reduced enzyme activity and inactivation respectively and have shown high prevalence in
African American populations (6, 8).
3
Gamma-Glutamyl Carboxylation (GGCX) located at chromosome 2p12 region, is responsible
for the anticoagulation process of vitamin K cycle (9) and rs11676382 (16025G>C) variant
correlates with reduced warfarin dose and seen in Caucasians (9-11).
Cytochrome P450 family 4, subfamily F, polypeptide 2 (CYP4F2) located at chromosome
19p13 region, is responsible in the vitamin K1 oxidase activity (also in Vitamin E) and
rs2108622(C>T) (V433M) variant is corresponds to increase warfarin dose and seen in
Caucasians (3, 12, 13).
By using an individual’s genotypes for these polymorphisms, therapeutic dose of Warfarin
can be estimated and personalized. The detection of variants at two genetic loci, which
explain as much as 50% of dose variability and wide differences in individual dose, has
further generated significance in warfarin pharmacogenetics with the promise of improved
therapy (4, 6, 14). Although testing for CYP2C9*2 and CYP2C9*3 for warfarin therapy has
been done in Sri Lanka, CYP4F2 V433M and GGCX have not been tested.
The objective of this study is to design and implement a genotyping assay for
CYP2C9*2,*3,*5,*6, CYP4F2 and GGCX polymorphisms to use as estimates for
individualized Warfarin dosage.
MATERIALS AND METHODS
Clinical samples of pre-purified hgDNA from 20 de-identified patients were obtain from our
clinic for the study to optimize the assay. A tetra primer multiplexed allele specific PCR
assay to genotype CYP2C9*2, CYP2C9*3, CYP2C9*5, CYP2C9*6, CYP4F2 V433M and
GGCX variants was designed. These novel primers have specific 3’-ends, which were
manipulated to distinguish single nucleotide change at the specific locus during PCR
amplification.
The
reference
sequence
was
taken
from
SNPedia
4
(http://www.snpedia.com/index.php/SNPedia)
and
dbSNP
(http://www.ncbi.nlm.nih.gov/SNP/) data bases. The tetra primers were designed using
Primer1 (http://primer1.soton.ac.uk/primer1.html).
Six 25µl Master Mixers were made including one variant each to obtained more accurate
results, CYP2C9*2, CYP2C9*6, CYP2C9*3, CYP4F2 V433M, CYP2C9*5, GGCX. Master
mixer for the optimized protocol comprises ddH2O 13.9µL, 12.5µL, 13.6µL, 14.1µL,
12.7µL, 12.7µL respectively, 5X Buffer(Promega) 5 µL, MgCl2(25mM)(Promega) 1.5µL,
dNTP mix(10mM)(Promega) 1.0µL, 0.2 µL of (5u/µL)Taq Polymerase each, 2.0 µL of DNA
and different volumes of primers as mentioned below in Table 1 and PCR amplification was
performed using ABI 2720 and ABI 9800 PCR machines (Applied Biosystems, Foster City)
with an initial denaturation at 95 °C for 10mins, followed by total 35 cycles which compiled
of denaturation at 95 °C for 30sec
and extension at 72 °C for 30sec with annealing
temperatures of 53 °C, 57 °C and 60 °C respectively at the final extension of 72 °C for 7mins.
The PCR product was analyzed by electrophoresis of 3% Agarose gel stained in ethidium
bromide at 80 V for 1hr.
According to previously published articles, the computationally designed primers were
optimized and troubleshooting was performed to reduce the shortcomings and to obtain
optimal results (15-18). The validation of this novel Multiplex AS PCR assay was achieved
by Sanger sequencing using an ABI 3030 DNA sequencer (Applied Biosystems, Foster city)
by sequencing the outer primers of all six variants and used it as the reference DNA.
The variants were multiplexed to reduce the laboriousness. Two variants in a single tube were
multiplexed; Mix 1: CYP2C9*2 and CYP2C9*6, Mix 2:CYP2C9*3 and CYP4F2, Mix
3:CYP2C9*5 and GGCX to obtain clarity and the resolutions of the bands. The concentrations
of Taq polymerase, MgCl2 and dNTP had distinct effects on the specificity and relative yield
5
of the PCR products. Although increasing the concentration in the PCR mix increased the
intensity of our desired bands, it also resulted in more nonspecific backgrounds. To obtain the
optimal annealing temperature, MgCl2 and dNTP, a gradient PCR was performed. Primer
concentration of each variant was also changed to obtain a visible band.
RESULTS
This newly designed genotyping assay was obtained by primer design, in vitro testing and a
series of optimizations. A set of 24 primers and specific PCR reaction conditions were
evaluated for accurate and reproducible genotyping of clinical samples. The gel
electropherogram and the bands which were obtained are shown in figure 1.
Products of each of the 6 variants constituting this assay were successfully amplified
individually, and samples were sequenced to verify the results. The optimum assay
parameters for the multiplex PCR for CYP2C9*2,*3,*5,*6, CYP4F2 and GGCX were 3
mixtures of 2.0mmol/l of MgCl2, 0.5mmol/l dNTP, 0.4mmol/l of 5U Taq DNA polymerase,
the same primer volumes mentioned below (Table.1) in a final volume of 25µl each using
57 °C as the annealing temperature.
The multiplexed method was found not to be reproducible among some variants but specific
when tested against 20 DNA samples as well as with single variant PCR analysis. Except for
CYP2C9*5, rest of the variants gave the expected bands when multiplexed. In addition,
except for CYP2C9*2 allele showing two positive patients other alleles were found to be
normal among the sample cohort. This was further confirmed with sequencing data.
6
Table 1. Primer sequences of CYP2C9, CYP4F2 and GGCX variants.
Primer
Sequence
CYP2C9*2
Fw:
5’ - CTGGGATCTCCCTCCTAGTTTCGTTT -3’
COM
Rv :
5’- ATTCCCTTGGCTCTCAGCTTCAAAC -3’
COM
Fw:
5’ - GGAAGAGGAGCATTGAGGCCC - 3’
WT
Rv:
5’ - CGGGCTTCCTCTTGAACCCA -3’
MUT
CYP2C9*6
Fw:
5’- AACCAGAGCTTGGTATATGGTATGTATGCTT -3’
COM
Rv :
5’- TGATCTCCCCTTTATCATTTTTATTGTGTC -3’
COM
Fw:
5’- TGATTGCTTCCTGATGAAAATGGATAG -3’
MUT
5’Rv: WT GCAGTCACATAACTAAGCTTTTGTTTACATTTTAACT3’
CYP2C9*3
Fw:
5’- TGTGCCATTTTTCTCCTTTTCCAT -3’
COM
Rv :
5’- TGAGTTATGCACTTCTCTCACCCG -3’
COM
Fw:
5’ – GCACGAGGTCCAGAGATGCA -3’
WT
Rv:
5’- CTGGTGGGGAGAAGGTCGAG -3’
MUT
CYP4F2 V433M
Fw:
5’- CTCTGGGTCAAAGCGAAAGG -3’
COM
Rv :
5’- TTTGCCCTTCCTGACCATGT -3’
COM
Fw:
5’- CACCTCAGGG TCCGGCCAGAC -3’
WT
Rv:
5’- GAACCCATCACAACCCAGGTA -3’
MUT
CYP2C9*5
Fw:
5’- AGATTGAACGTGTGATTGGCAGAA -3’
COM
Rv :
5’-TTGGGGACTTCGAAAACATGGA -3’
COM
Fw:
5’- CACGAGGTCCAGAGATACATTCAC -3’
WT
Rv:
5’- GCAGGCTGGTGGGGAGATGC-3’
MUT
GGCX
Fw:
5’- CAGAACAAGAAAGCAGGCCATCAGA -3’
COM
Rv :
5’- AGCCAACACCTCTGGTTCAGACCTT -3’
COM
Fw:WT 5’- CCCCAGGGGAAAGTTACCATGC -3’
Rv:
5’- TTTGTCATTGCCATCATATGTTGGCTAC -3’
MUT
Location
Fragment
Size(bp)
264-289
Calculated
Tm(°C)
Primer
Concentration
(µL)
66
0.40
66
0.40
344
607-583
381-401
228
66
0.30
420-401
157
66
0.30
65
0.60
65
0.60
8-38
478
405-456
231-257
256
65
0.80
293-257
286
65
0.80
64
0.40
64
0.40
240-263
343
582-559
382-401
202
64
0.40
420-401
181
64
0.40
66
0.40
57
0.40
279-298
483
743-742
581-501
281
58
0.20
521-501
242
59
0.20
62
0.60
69
0.60
141-171
222
369-348
228-251
143
65
0.60
270-251
123
65
0.80
67
060
67
0.60
255-279
323
578-554
380-401
200
67
0.60
428-401
174
67
0.80
7
Figure 1. The PCR assay: (A) T-ARMS PCR assay using single variants; Lane 1: 100bp
ladder, Lane 2: CYP2C9*2, 344bp (control) and 220bp (wild) bands, Lane 3: CYP2C9*6,
478bp (control) and 256bp (wild) bands, Lane 4: CYP2C9*3, 343bp (control) and 202bp
(wild) bands, Lane 5: 100bp ladder, Lane 6: CTP4F2, 483bp (control) and 282bp (wild)
bands, Lane 7: GGCX, 324bp (control) and 200bp (wild) bands, Lane 8: CYP2C9*5, 222bp
(control) and 143bp (wild) bands, Lane 9: Blank (B) Multiplexed PCR assay using two
variants; Lane 1: 100bp ladder, Lane 2: Mix 1- CYP2C9*2, CYP2C9*6, Lane 3: Mix 2CYP2C9*3, CYP4F2, Lane 4: GGCX
DISCUSSION
In response to the prevalence of these variants and the association of these with warfarin
dosage, we developed a simple CYP2C9, CYP4F2 and GGCX T-ARMS PCR genotyping
assay to implement in our clinical laboratory. A method of 24 primers was designed to
explicitly determine a patient’s genotype at the CYP2C9 *2, *3, *5, *6, CYP4F2 and GGCX
SNP sites in a single variant tetra primer multiplexed PCR reaction. We genotyped 20
genomic DNA samples and validated the method by sequencing.
Allele specific PCR is a cost effective, time saving PCR technique and the multiplexing
approach is a promising method to overcome the shortcomings of current single PCR
reactions and to increase the diagnostic capacity of PCR (15). In multiplex PCR, more than
one target variant would be amplified by including more than one pair of primers in one PCR
8
reaction (15, 16). The success of a multiplex PCR is governed by the PCR cycling conditions
and PCR mix. Sub-optimal conditions of any PCR reagents would result in problems such as
failure to amplify, amplification of nonspecific products, products of wrong size, high
molecular weight smears or primer dimers. Modifications of parameters are known to
contribute to the primer-template reliability and primer extension has to be performed.
We designed our primers specifically to match the polymorphic sites. Mismatches of the base
at the 3’ end of a primer can cause PCR failures. The T-ARMS PCR primer sets used in this
research were found to be PCR compatible as no significant primer dimers or nonspecific
bands were observed. An optimum PCR condition would increase the specificity of methods
developed. The presence of nonspecific bands and smearing backgrounds obtained from our
initial PCR condition for the single variants T-ARMS PCR was reduced by optimizing the
concentrations for magnesium, dNTP as well as altering the annealing temperature; but was
not the same with two variants multiplexed PCR method. The primers concentration was
found to be a sensitive parameter that determined specificity and the visibility of the band.
Warfarin is the leading oral anticoagulant for reducing thromboembolic events that often
gives rise to stroke, deep vein thrombosis, pulmonary embolism or serious coronary
malfunctions worldwide. In addition, warfarin has been identified as the second leading cause
of drug-related emergency room visits (17). Warfarin has a narrow therapeutic index and the
maintenance dose is difficult to predict ranging from 0.5–60 mg/day with an average dose of
∼4–6mg/day (28 to 42 mg/wk) (18). Many studies have shown that a genetic determinant of
interindividual warfarin dose variability is associated with the genotypes of several single
nucleotide polymorphisms. These variants are involved in warfarin pharmacokinetics and
pharmacodynamics, respectively, and when combined with other clinical and environmental
factors, leads approximately 50% of interindividual warfarin dose variations (19).
9
Gene dosage can affect the metabolic activity of CYP2C9 and hence the metabolism of
warfarin. Patients who have one copy of the CYP2C9*2 are slow metabolizers of S-warfarin;
patients who are homozygous for CYP2C9*2 or who carry at least 1 copy of the CYP2C9*3,
CYP2C9*5 or CYP2C9*6 SNP are very slow metabolizers. Previous studies indicate that
patients carrying the CYP2C9*2 and CYP2C9*3 have shown to require more time to achieve
stable dosing to require lower maintenance dose of warfarin (5). Carriers of CYP4F2 V433M
(rs2108622 C>T) have reduced function, resulting in approximately an 8% increase in
warfarin dose (20). GGCX rs11676382 is associated with a 6% reduced warfarin dose per G
allele (10). The pharmacogenomic algorithm produced significantly better dose estimates,
and provides a robust basis for the genetically informed dose estimation for patients who
require warfarin (21). By genotyping these alleles and entering the data to the
warfarindosing.org web site (http://www.warfarindosing.org/Source/Home.aspx), the dose
alteration would be even more accurate.
Our methods may require further troubleshooting and optimization to get an optimal PCR
condition and to obtain reproducibility of the two variants multiplexed PCR method as this
would be useful for our routine laboratory practice. Even though the single variant T-ARMS
multiplexed PCR procedure is relatively time consuming, the results can be obtained within 3
hrs hence it can be implemented in the clinical laboratory. As the genetic variants of
CYP2C9, CYP4F2 and GGCX genes shows a significant association with the therapeutic dose
of warfarin, it is important to genotype these variants among the patients who require
warfarin. By implementing this genotyping assay, it would be medically and economically
beneficial, cost effective pharmacogenomic platforms so that the therapeutic dose can be
personalized in patients.
10
REFERENCES
1.
Kamali F, Wynne H. Pharmacogenetics of warfarin. Annu Rev Med. 2010;61:63-75.
2.
Krishna Kumar D, Shewade DG, Loriot MA, Beaune P, Balachander J, Sai Chandran
BV, Adithan C. Effect of CYP2C9, VKORC1, CYP4F2 and GGCX genetic variants on
warfarin maintenance dose and explicating a new pharmacogenetic algorithm in South
Indian population. Eur J Clin Pharmacol. 2014 Jan;70(1):47-56.
3.
LIng CS. Clinical Applications of Pharmacogenomics of Warfarin. Singapore: National
University; 2012.
4.
Blann A, Hewitt J, Siddiqui F, Bareford D. Racial background is a determinant of
average warfarin dose required to maintain the INR between 2.0 and 3.0. Br J Haematol.
1999 Oct;107(1):207-9.
5.
Xie HG, Prasad HC, Kim RB, Stein CM. CYP2C9 allelic variants: ethnic distribution
and functional significance. Adv Drug Deliv Rev. 2002 Nov 18;54(10):1257-70.
6.
Carlquist J, Horne B, Mower C, Park J, Huntinghouse J, McKinney J, Muhlestein J,
Anderson J. An evaluation of nine genetic variants related to metabolism and mechanism
of action of warfarin as applied to stable dose prediction. Journal of Thrombosis and
Thrombolysis. 2010 2010/10/01;30(3):358-64.
7.
Sim SC, Ingelman-Sundberg M. The Human Cytochrome P450 (CYP) Allele
Nomenclature website: a peer-reviewed database of CYP variants and their associated
effects. Hum Genomics. 2010 Apr;4(4):278-81.
8.
Ali ZK, Kim RJ, Ysla FM. CYP2C9 polymorphisms: considerations in NSAID therapy.
Curr Opin Drug Discov Devel. 2009 Jan;12(1):108-14.
9.
Rieder MJ, Reiner AP, Rettie AE. Gamma-glutamyl carboxylase (GGCX) tagSNPs have
limited utility for predicting warfarin maintenance dose. J Thromb Haemost. 2007
Nov;5(11):2227-34.
11
10.
King CR, Deych E, Milligan P, Eby C, Lenzini P, Grice G, Porche-Sorbet RM, Ridker
PM, Gage BF. Gamma-glutamyl carboxylase and its influence on warfarin dose.
Thromb Haemost. 2010 Oct;104(4):750-4.
11.
Wadelius M, Chen LY, Downes K, Ghori J, Hunt S, Eriksson N, Wallerman O, Melhus
H, Wadelius C, Bentley D, Deloukas P. Common VKORC1 and GGCX
polymorphisms associated with warfarin dose. Pharmacogenomics J. 2005;5(4):262-70.
12.
Cen HJ, Zeng WT, Leng XY, Huang M, Chen X, Li JL, Huang ZY, Bi HC, Wang XD,
He YL, He F, Zhou RN, Zheng QS, Zhao LZ. CYP4F2 rs2108622: a minor significant
genetic factor of warfarin dose in Han Chinese patients with mechanical heart valve
replacement. Br J Clin Pharmacol. 2010;70(2):234-40.
13.
McDonald MG, Rieder MJ, Nakano M, Hsia CK, Rettie AE. CYP4F2 is a vitamin K1
oxidase: An explanation for altered warfarin dose in carriers of the V433M variant. Mol
Pharmacol. 2009 Jun;75(6):1337-46.
14.
Sconce EA, Khan TI, Wynne HA, Avery P, Monkhouse L, King BP, Wood P, Kesteven
P, Daly AK, Kamali F. The impact of CYP2C9 and VKORC1 genetic polymorphism
and patient characteristics upon warfarin dose requirements: proposal for a new dosing
regimen. Blood. 2005 Oct 1;106(7):2329-33.
15.
Zainuddin Z, Teh LK, Suhaimi AW, Salleh MZ, Ismail R. A simple method for the
detection of CYP2C9 polymorphisms: nested allele-specific multiplex polymerase
chain reaction. Clin Chim Acta. 2003 Oct;336(1-2):97-102.
16.
Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH. Multiplex PCR: critical
parameters and step-by-step protocol. Biotechniques. 1997 Sep;23(3):504-11.
17.
Poe BL, Haverstick DM, Landers JP. Warfarin genotyping in a single PCR reaction for
microchip electrophoresis. Clin Chem. 2012 Apr;58(4):725-31.
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18.
Linder MW, Looney S, Adams JE, 3rd, Johnson N, Antonino-Green D, Lacefield N,
Bukaveckas BL, Valdes R, Jr. Warfarin dose adjustments based on CYP2C9 genetic
polymorphisms. J Thromb Thrombolysis. 2002 Dec;14(3):227-32.
19.
Scott SA, Khasawneh R, Peter I, Kornreich R, Desnick RJ. Combined CYP2C9,
VKORC1
and
CYP4F2
frequencies
among
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and
ethnic
groups.
Pharmacogenomics. 2010 Jun;11(6):781-91
20.
Caldwell MD, Awad T, Johnson JA, Gage BF, Falkowski M, Gardina P, Hubbard J,
Turpaz Y, Langaee TY, Eby C, King CR, Brower A, Schmelzer JR, Glurich I, Vidaillet
HJ, Yale SH, Qi Zhang K, Berg RL, Burmester JK. CYP4F2 genetic variant alters
required warfarin dose. Blood. 2008 Apr 15;111(8):4106-12.
21.
Estimation of the Warfarin Dose with Clinical and Pharmacogenetic Data. New
England Journal of Medicine. 2009;360(8):753-64.
13
SOP
: Warfarin panel Genotyping
Title
: Detection of CYP2C9,CYP4F2,GGCX variant alleles
Last Revised
: April 2014
Test
: Molecular Genetic Test
Purpose
: Detect the presence of CYP2C9*2,*3,*5,*6, CYP4F2 V433M and GGCX variants to
predict the effect of Warfarin drug metabolism.
Method
: Multiplex Allele- Specific PCR (AS-PCR)
1. DNA extraction
2. Multiplex allele specific PCR
3. Agarose gel electrophoresis
Step 1
Primer reconstitution (SOP003).
PRIMER
SEQUENCE
CYP2C9*2
CYP2C9*3
GGCX
CYP4F2
V433M
CYP2C9*6
818delA
CYP2C9*5
Step 1
F(C)
R(T)
C[F]
C[R]
F(A)
5’ - GGAAGAGGAGCATTGAGGCCC - 3’
5’ - CGGGCTTCCTCTTGAACCCA -3’
5’ - CTGGGATCTCCCTCCTAGTTTCGTTT -3’
5’- ATTCCCTTGGCTCTCAGCTTCAAAC -3’
5’ – GCACGAGGTCCAGAGATGCA -3’
R(C)
C[F]
C[R]
F(C)
R(G)
C[F]
C[R]
F(C)
5’- CTGGTGGGGAGAAGGTCGAG -3’
5’- TGTGCCATTTTTCTCCTTTTCCAT -3’
5’- TGAGTTATGCACTTCTCTCACCCG -3’
5’- CCCCAGGGGAAAGTTACCATGC -3’
5’- TTTGTCATTGCCATCATATGTTGGCTAC -3’
5’- CAGAACAAGAAAGCAGGCCATCAGA -3’
5’- AGCCAACACCTCTGGTTCAGACCTT -3’
5’- CACCTCAGGG TCCGGCCAGAC -3’
R(T)
C [F]
C[R]
F(G)
R(A)
C[F]
C[R]
F(C)
R(G)
C[F]
C[R]
5’- GAACCCATCACAACCCAGGTA -3’
5’- CTCTGGGTCAAAGCGAAAGG -3’
5’- TTTGCCCTTCCTGACCATGT -3’
5’- TGATTGCTTCCTGATGAAAATGGATAG -3’
5’- GCAGTCACATAACTAAGCTTTTGTTTACATTTTAACT -3’
5’- AACCAGAGCTTGGTATATGGTATGTATGCTT -3’
5’- TGATCTCCCCTTTATCATTTTTATTGTGTC -3’
5’- CACGAGGTCCAGAGATACATTCAC -3’
5’- GCAGGCTGGTGGGGAGATGC-3’
5’- AGATTGAACGTGTGATTGGCAGAA -3’
5’-TTGGGGACTTCGAAAACATGGA -3’
: DNA extraction using peripheral blood leucocytes (Ref.SOP001/SOP002)
1
Step 2
Set-up PCR:25µL Master mix contains;
Master Mix
ddH2O
1
13.9 µL
2
12.5 µL
3
13.6 µL
4
14.1 µL
5
12.7 µL
5x buffer
5.0 µL
5.0 µL
5.0 µL
5.0 µL
5.0 µL
MgCl2(25mM)
1.5 µL
1.5 µL
1.5 µL
1.5 µL
1.5 µL
dNTPmix(10mM)
CYP2C9*2 F(C)
1.0 µL
0.3 µL
1.0 µL
1.0 µL
1.0 µL
1.0 µL
1.
2.
CYP2C9*2 R(T)
0.3 µL
3.
CYP2C9*2 C[F]
0.4µL
4.
CYP2C9*2 C[R]
0.4µL
5.
CYP2C9*6 818delA F(G)
0.8 µL
6.
CYP2C9*6 818delA R(A)
0.8 µL
7.
CYP2C9*6 818delA C[F]
0.6µL
8.
CYP2C9*6 818delA C[R]
0.6µL
9.
10.
11.
12.
13.
CYP2C9*3 F(A)
CYP2C9*3 R(C)
CYP2C9*3 C[F]
CYP2C9*3C[R]
CYP4F2 V433M F(C)
6
12.7
µL
5.0
µL
1.5
µL
1.0 µL
0.4 µL
0.4 µL
0.4 µL
0.4 µL
0.2 µL
14. CYP4F2 V433M R(T)
0.2 µL
15. CYP4F2 V433M C[F]
0.4µL
16. CYP4F2 V433M C[R]
0.4µL
17. GGCX F(C)
18. GGCX R(G)
0.8 µL
0.6µL
GGCX C[F]
0.6µL
GGCX C[R]
0.6µL
CYP2C9*5 F(C)
0.6 µL
CYP2C9*5 R(G)
0.8µL
CYP2C9*5 C[F]
0.6µL
CYP2C9*5 C[R]
0.6µL
Taq
DNA
Total
0.2
2.0
25µL
0.2
2.0
25µL
0.2
2.0
25µL
0.2
2.0
25µL
0.2
2.0
25µL
0.2
2.0
25µL
2
Step 3
Run PCR
PCR cycle conditions:
Initial denaturation at 95˚c for 10 mins.
Denaturation at 95 ˚c for 30 sec.
Annealing at 53 ˚c for 30 sec.
5 cycles
Extension at 72 ˚c for 30 sec.
Denaturation at 95 ˚c for 30 sec.
Annealing at 57 ˚c for 30 sec.
15 cycles
35 Cycles
Extension at 72 ˚c for 30 sec
Denaturation at 95 ˚c for 30 sec.
Annealing at 60 ˚c for 30 sec.
15 cycles
Extension at 72 ˚c for 30 sec
Final extension at 72 ˚c for 7 min.
Cooling at 4 ˚c ∞
Step 4
: Analysis by gel electrophoresis (Ref:SOP004)
Run 10µl of PCR product in 3% gel at 80V for 1hr.
Gel
:
Interpretation
3
Allele
CYP2C9*2 (C)
CYP2C9*2 (T)
CYP2C9*2 outer
CYP2C9*6 (G)
CYP2C9*6 (A)
CYP2C9*6 outer
CYP2C9*3 (A)
CYP2C9*3 (C)
CYP2C9*3 outer
CYP4F2 (C)
CYP4F2 (T)
CYP4F2 outer
GGCX (C)
GGCX (G)
GGCX outer
CYP2C9*5 (C)
CYP2C9*5 (G)
CYP2C9*5 outer
Reference
Bp size
220bp
157bp
344bp
256bp
286bp
478bp
202bp
181bp
343bp
281bp
242bp
483bp
200bp
174bp
324bp
143bp
123bp
222bp
: -
4
CYP2C9, CYP4F2, GGCX Genotyping Assay for to predict
Warfarin Maintenance Dose
Human Genetics Unit, Faculty of Medicine, University of Colombo
Warfarin (brand name Coumadin) is the most
country, they are not tested. Non genetic factors and
widely prescribed oral anticoagulant drug in the
variants in other genes involving the drug metabolism are
world which is used for the prevention and
not measured by this assay. Results of this test should be
treatment of arterial and venous thrombo-
interpreted in the context of clinical presentation and in
embolic diseases including Deep Vein Thrombosis
consultation with a licensed geneticist and/or pharmacist.
(DVT), Pulmonary Embolism (PE), Ischemic
Stroke, Myocardial Infarction (MI), and Arterial
Testing Methodology:
Fibrillation.
constricted
This test is performed by DNA amplification using a tetra
therapeutic index and a wide inter-individual
primer Amplification-refractory mutation system polymerase
unpredictability in dose requirement and at risk
chain reaction (T-ARMS PCR) followed by gel electrophoresis
with under dosing or overdosing, respectively.
and viewing under UV irradiation.
Single
Warfarin
nucleotide
has
a
polymorphisms
in
the
cytochrome P450 2C9 (CYP2C9), GGCX and
Variant Alleles Tested:
CYP4F2 V433M genes have a significant effect on
*2 (c.430C>T) - Reduced activity
warfarin dose requirement. CYP2C9 variants such
*3 (c.1075A>C) - Reduced activity
as CYP2C9*2 (rs1799853, 430C>T), CYP2C9*3
*5 (c.1080C>G) - Reduced activity
(rs1057910, 1075A>C) variants lead to reduced
*6 (c.818delA) - No activity
enzyme activity and more prevalent in Asian and
GGCX: c.16025G>C - Reduced activity
Caucasian population and CYP2C9*5 (rs28371686,
CYP4F2 V433M: rs2108622 C>T – Increased activity
1080C>G), CYP2C9*6 (rs9332131, 818delA) leads
to reduced enzyme activity and inactivation
respectively and have shown high prevalence in
Specimen Requirements:

Peripheral blood: Collected in EDTA (purple top) tube.
African American population. Gamma-Glutamyl
Carboxylation (GGCX) variant is correlated with
reduced warfarin dose and seen in Caucasians.
Cytochrome
P450
family
4,
subfamily
Adults: 3cc

Handling: Room Temp- specimen processed within 72
hours
F,
polypeptide 2 (CYP4F2) rs2108622 (1297C>T)
Turnaround Time:
(V433M) variant correspond to increased warfarin
14 days
dose and seen in Caucasians.
Reasons for Referral:
 Patients starting initial treatment with Warfarin.
 Patients already treated with warfarin that experiencing
difficulties
in
achieving
therapeutic
International
normalize ratio (INR).
 Results of this test can be used in available algorithms
to determine the appropriate Warfarin dosing (www.
Warfaindosing.org)
Limitations:
Only the most common alleles are targeted by this assay,
due to the lower prevalence of the other alleles in the
Perforating Laboratory:
Human Genetics Unit
Faculty of Medicine
University of Colombo
Kynsey Road, Colombo 8, Sri Lanka
Phone (94-011) 2695 300, 2689 545 (Direct)
Fax (94-011) 2689979
[email protected]
http://www.hgucolombo.org
References:
Kamali F, Wynne H. Pharmacogenetics of warfarin. Annu Rev
Med. 2010;61:63-75.
Krishna Kumar D, Shewade DG, Loriot MA, Beaune P,
Balachander J, Sai Chandran BV, Adithan C. Effect of CYP2C9,
VKORC1, CYP4F2 and GGCX genetic variants on warfarin
maintenance dose and explicating a new pharmacogenetic
algorithm in South Indian population. Eur J Clin Pharmacol.
2014 Jan;70(1):47-56.
Human Genetics Unit
Faculty of Medicine
University of Colombo
Kynsey Road, Colombo 8, Sri Lanka
Phone (94-011) 2695 300, 2689 545 (Direct)
Fax (94-011) 2689979
[email protected]
http://www.hgucolombo.org
Confidential Molecular Genetic Laboratory Test Report
Patient Identification:
Lab Reference:
Indication:
Material Tested:
Test:
Analysis Performed:
Name:
Result:
NAME OF GENE: CYP2C9
Variants tested
rs1799853 (CYP2C9*2)
rs1057910 (CYP2C9*3)
rs28371686 (CYP2C9*5)
rs9332131 (CYP2C9*6)
NAME OF GENE: CYP4F2 (433V>M)
Variants tested
rs2108622
NAME OF GENE: GGCX (16025G>C)
Variants tested
rs1167638
Remaks:
Date :
Age:
Sex:
EDTA Blood
Warfarin panel genotyping
The following mutations were genotyped by T-ARMS PCR:
CYP2C9*2 ( rs1799853, 430C>T)
CYP2C9*3 (rs1057910, 1075A>C)
CYP2C9*5 (rs28371686, 1080C>G)
CYP2C9*6 (rs9332131, 818delA)
CYP4F2 (rs2108622 C>T)
GGCX
(rs11676382, 16025G>C)
LOCATION OF GENE: 10q24
RESULT
CC
AA
CC
AA
LOCATION OF GENE: 19p13
RESULT
CC
LOCATION OF GENE: 2p12
RESULT
GG
Based on this individual’s Combined Genetic Result: CYP2C9*2 ,*3, *5, *6,
CYP4F2-CC, GGCX-GG, warfarin Dose should be adjusted according to the
www.warfarindosing.org.
Test Limitations: There may be other variants in the CYP2C9 gene ,
VKORC1 gene or the CYP4F2 gene that are not included in this test, that
influence the response to warfarin.
Prof. Vajira H. W. Dissanayake MBBS, PhD
Medical Geneticist
Analysis Performed by:…………………………
Analysis Requested by:
Because of their complexity and their potential implications for other family members, all genetic tests should be
accompanied by genetic counseling.
Prof. Rohan W. Jayasekara MBBS (Ceylon), PhD (Newcastle), C.Biol., MSB (London) – Medical Geneticist and Director
Prof. Vajira H. W. Dissanayake MBBS (Colombo), PhD (Nottingham) – Medical Geneticist
DESIGN AND IMPLEMENTATION OF A
TETRA PRIMER AMPLIFICATION
REFRACTORY MUTATION SYSTEM
POLYMERASE CHAIN REACTION
(ARMS/PCR) GENOTYPING ASSAY
FOR CYP2C19*2 AND CYP2C19*17
POLYMORPHISMS
ABSTRACT
Introduction: The cytochrome P450 2C19 (CYP2C19) is important for the metabolism of
several therapeutic agents such as proton pump inhibitors (PPIs), anticonvulsants, antiulcer,
antiseizure and antidepressants drugs such as omeprazole, tamoxifen, clopidogrel, fluoxetine,
lansoprazole, S-mephenytoin, tolbutamide, voriconazole and diazepam. CYP2C19*2, the
most common variant allele of CYP2C19 alters the reading frame of mRNA from amino acid
215, and produces a stop codon 20 bp downstream, leading to a truncated protein. This allele
is associated with a distinct decrease in platelet response. CYP2C19*17 is a recently
identified variant where the carriers of this variant were found to have a significantly lower
Area under the curve (AUC), suggesting that this would give rise to an extensive metabolizer
phenotype for CYP2C19. Testing for CYP2C19*2 and CYP2C9*17 has never been conducted
in Sri Lanka.
Objective: Objective of this study was to design and implement a genotyping assay for
CYP2C19*2 and CYP2C19*17 polymorphisms.
Materials and Methods: Clinical samples of pre-purified hgDNA from 32 de-identified
patients were used to implement and optimize a T-ARMS multiplexed PCR method. These
primers were obtained from a previously published article and troubleshooting, optimization
and sequencing were performed to obtain the optimal results.
Results: The desired specific bands were obtained by many optimizations and
troubleshooting as the reported article had errors in PCR protocol and PCR conditions.
Discussion and Conclusion: The optimal PCR conditions and parameters were obtained by
troubleshooting. Further optimizations are required to achieve substantial optimal parameters.
Additionally genotyping CYP2C19*3 variant should be considered as this would be useful for
1
our routine laboratory use. As the genetic variants of CYP2C19*2 and CYP2C19*17 variants
shows significant association with the therapeutic dose of approximately 15% of all
prescribed mainstream drugs, it is important to genotype these variants among the patient
population. By implementing this genotyping assay, it would be a medically and
economically beneficial, cost effective pharmacogenomic platform to be used as an aid in
determining personalized therapeutic strategies.
KEY WORDS: CYP2C19*2, CYP2C19*17, T-ARMS multiplexed PCR, Clopidogrel,
Tamoxifen
2
INTRODUCTION
The cytochrome P450 2C19 (CYP2C19) is important for the metabolism of several
therapeutic agents such as proton pump inhibitors (PPIs), anticonvulsants, antiulcer,
antiseizure and antidepressants drugs such as omeprazole, tamoxifen, clopidogrel fluoxetine,
lansoprazole, S-mephenytoin, tolbutamide, voriconazole and diazepam (1). The therapeutic
effect of CYP2C19 substrates can be attributed to the genetic polymorphisms of CYP2C19
gene. The CYP2C19 gene maps onto chromosome 10 (10q24.1-q24.3), and encodes a 490amino-acid protein. Approximately 27 variant alleles in the CYP2C19 gene have been
identified (CYP2C19*2 to *27) (http://www.imm.ki.se/CYPalleles, access date: 20 February
2010). Large inter-individual differences have been seen in the metabolism of these drugs in
vivo, and individuals can be divided in to normal, extensive metabolizer, (EM), intermediate
metabolizer (IM), poor metabolizer (PM), and ultra rapid metabolizers (UM) (2). The ‘poor
metabolizer’ phenotype of these variants is common, occurring worldwide at 0.02-0.05% in
Caucasians and 0.18-0.23% in Asians (3, 4). Polymorphisms; CYP2C19*2 CYP2C19*17
exists, with approximately 3–5% of Caucasian and 15–20% of Asian populations being poor
metabolizers with no CYP2C19 function (5, 6) which may reduce the efficacy of clopidogrel.
CYP2C19*2 (rs4244285), the most common variant allele of CYP2C19, is a single base pair
681G>A mutation on exon 5, leading to an aberrant splice site (7, 8). This change alters the
reading frame of mRNA from amino acid 215, and produces a stop codon 20 bp downstream,
leading to a truncated protein. This allele is associated with a distinct decrease in platelet
response to clopidogrel in unsteady patients (9) and an impaired prediction in clopidogreltreated patients. The relative frequency of the CYP2C19*2 variant among the south Indian
population was found to be 35% (10).
3
CYP2C19*17 (rs12248560) is a recently identified variant in the CYP2C19 gene. The
CYP2C19*17 allele is characterized by two SNPs in the 5′-flanking region (g.-3402C > T and
g.-806C > T) of the gene. The two polymorphisms are in complete linkage disequilibrium
with each other. Carriers of this variant were found to have a significantly lower Area under
the curve (AUC) of omeprazole, suggesting that this would give rise to an extensive
metabolizer phenotype for CYP2C19 (8). The variant allelic frequency of CYP2C19*17 is
reported to be 2-4% and 18-20% in Asian and Caucasian (11) populations respectively.
Using an individual’s genotypes, therapeutic dose of some of the above mentioned drugs can
be estimated and personalized. The detection of variants at two genetic loci, would explain
the dose variability and wide differences in individual dose. Testing for CYP2C19*2 and
CYP2C9*17 have never been carried out in Sri Lanka.
The objective of this study was to design and implement a genotyping assay for CYP2C19*2
and CYP2C19*17 to assess personalized relative medicine. The alleles were chosen
depending upon their prevalence and clinical benefits.
MATERIALS AND METHODS
Clinical samples of pre-purified hgDNA from 32 de-identified patients were obtained from
our clinic for the study to optimize the assay. A tetra primer multiplexed allele specific PCR
assay to genotype CYP2C19*2, CYP2C19*17 variants was designed. These primers were
obtained following a procedure from a previously published article by Cuisset T et al. (12).
The
reference
sequence
was
(http://www.snpedia.com/index.php/SNPedia)
taken
from
SNPedia
and
dbSNP
(http://www.ncbi.nlm.nih.gov/SNP/) data bases. The accuracy of these pre-designed primers
were
checked
using
EMBOSS
(www.ebi.ac.uk/Tools/emboss/)
and
UCSC
(https://genome.ucsc.edu/) web sites.
4
We used a newly optimized protocol consisting two master mixes of 25µl including one
variant each. It comprised of ddH2O 10.3µL, 12.5µL respectively, 5X Buffer(Promega) 5µL,
MgCl2(25mM)(Promega) 1.5µL, dNTP mix (10mM)(Promega) 1.0µL, 0.2 µL of (5u/µL)Taq
polymerase each, 2.0 µL of DNA and different volumes of primers as mentioned below. PCR
amplification was performed using ABI 2720 and ABI 9800 PCR machines (Applied
Biosystems, Foster City) with an initial hot start at 95 °C for 10mins, followed by total 35
cycles which comprised of denaturation at 95 °C for 30sec, and extension at 72 °C for 30sec
with annealing temperatures of 53 °C, 57 °C and 60 °C respectively at the final extension of
72 °C for 7mins. The PCR product was analyzed by electrophoresis of 3% Agarose gel
stained in ethidium bromide at 80 V for 1hr.
To validate this novel multiplex AS PCR assay, we performed Sanger sequencing using ABI
3030 DNA sequencer (Applied Biosystems, Foster City) of the outer primers of the two
variants and used that sample as the reference DNA.
Table 1. Primer sequences of CYP2C19*2 and CYP2C19*17 variants.
Primer
Sequence
Location
Fw:
COM
Rv :
COM
Fw:
WT
5’ - CAG AGC TTG GCA TAT TGT ATC -3’
392-412
Rv: MUT
Fragment
Size(bp)
Calculated
Tm(°C)
Primer
Concentration
(µL)
65.3
1.50
67.1
1.50
CYP2C19*2
291
5’- TAT CGC AAG CAG TCA CATAAC -3’
662-681
5’ - ACT ATC ATT GAT TAT TTC CCG - 3’
481-501
201
62.2
1.00
5’ - GTA ATT TGT TAT GGG TTC CT -3’
501-520
128
62.2
1.00
64.3
1.00
64.7
0.60
CYP2C19*17
Fw:
5’- AAGAAG CCT TAG TTT CTC AAG -3’
COM
Rv :
5’- AAACACCTTTACCATTTAACCC -3’
COM
Rv:
5’- ATTA TCT CTT ACA TCA GAG ATG-3’
WT
Fw:
5’ -TGT CTT CTG TTC TCA AAG TA -3’
MUT
204-225
506
687-710
293-257
294
60.6
0.60
511-533
216
62.2
0.60
5
RESULTS
Results were obtained using in vitro testing and optimization. A set of 8 primers and specific
PCR reaction conditions were evaluated for accurate and reproducible genotyping of clinical
samples. The gel electropherogram and the bands which were obtained are shown in figure 1.
According to previously published reports, troubleshooting was performed to reduced the
shortcomings and acquire optimal results (13-16). We were unable to carry out the PCR cycle
of the pre designed PCR primers which were obtained from a previously published article
according to their protocol. Hence it had to be substantially modified and optimized for a new
PCR protocol. Products of each of the 2 variants constituting this assay were successfully
amplified individually, and samples were sequenced to verify the results.
The concentrations of Taq polymerase, MgCl2 and dNTP had different effects on the
specificity and relative yield of the PCR products. Altering the concentrations and other
parameters although it increased the intensity of our desired bands resulted in more
nonspecific backgrounds. To obtain the optimal annealing temperature, MgCl2 and dNTP, a
gradient PCR was performed. Only when the primer concentration of each variant was
increased than the mentioned parameters in the reported article, we obtained visible bands.
Though the reported article have mentioned the band sizes, when re-evaluated it was
different. The single variant T-ARMS multiplexed PCR was reproducible and specific when
tested against 32 DNA samples. The accuracy of the results was further confirmed with
sequencing data.
6
Figure 1. The PCR assay: (A) T-ARMS PCR assay using single variants; Lane 1: 100bp
ladder, Lane 2: CYP2C19*2, 291bp (outer) and 201bp (wild) bands Lane 3: CYP2C19*17,
506bp (control) and 294bp (wild) Lane 4: Blank (B) CYP2C19*2 genotype results; Lane 1:
50bp ladder, Lanes 2-7: heterozygous (291bp (outer), 201bp (wild) and 128bp (mutant)),
Lanes 8-9: Homozygous , Lane 10: reference DNA, Lane 11: Blank (C) CYP2C19*17
genotype results; Lane 1: 50bp ladder, Lanes 2,5,8,9: Heterozygous (506bp (control) and
294bp (wild) and 216 bp (mutant)), Lanes 3,4,6,7: Homozygous, Lane 11: reference DNA,
Lane 12: Blank.
DISCUSSION
With regard to the prevalence of these variants and the association of these with the
therapeutic dosage, we developed a simple CYP2C19*2 and CYP2C19*17 T-ARMS PCR
genotyping assay to implement in our clinical laboratory. A method of 8 primers was
obtained from the previously reported article yet performed the PCR according to a novel
protocol designed which would determine a patient’s genotype at CYP2C19 *2 and *17 sites
7
in a single variant tetra primer multiplexed PCR reaction. We genotyped 32 clinical patients
and validated by using the sequenced results.
Allele specific PCR is a cost effective, less laborious PCR technique. Sub-optimal conditions
of any PCR reagents would result in problems such as failure to amplify, amplification of
nonspecific products, products of wrong size, high molecular weight smears or primer
dimers. Modifications of parameters known to contribute to the primer-template reliability
and primer extension have to be performed.
The previously designed primers were checked using the online data bases to prove the
accuracy of alignments. The mentioned PCR protocol and conditions could not be achieved.
After many PCR optimizations to a new PCR protocol, the obtained band sizes were
different. When the band sizes were re-evaluated, we found that the obtained band sizes from
our protocol were the accurate band sizes expected for these variants. Due to this error, we
continued using our protocol with the use of the published and validated primers to perform
genotyping for CYP2C19. Visibility and clarity of the PCR bands were achieved only when
the primer volumes were increased than those mentioned. The mentioned PCR condition was
incapable of producing the CYP2C19*2 bands hence repeated troubleshooting was
performed. The presence of nonspecific bands and smearing backgrounds obtained from our
initial PCR condition for the single variants T-ARMS PCR was reduced by optimizing the
concentrations for magnesium, dNTP as well as annealing temperature. The primer
concentration was found to be a sensitive parameter that determined specificity and the
visibility of the band.
Inhibitors of CYP2C19 can be classified by their potency, such as: strong is when one that
causes at least a 5-fold increase in the plasma AUC values, or more than 80% decrease in
clearance. Moderate is when one that causes at least a 2-fold increase in the plasma AUC
8
values, or 50-80% decrease in clearance. Weak is when one that causes at least a 1.25-fold
but less than 2-fold increase in the plasma AUC values, or 20-50% decrease in clearance
(17).
Omeprazole, a proton pump inhibitor is hydroxylated by CYP2C19 to its primary metabolite
5-hydroxyomeprazole. It is used as a probe for setting up the genotype phenotype relationship
for CYP2C19 (18). Investigations on CYP2C19 effects on Omeprazole metabolism reported
that the metabolic ratio (Omeprazole/5-OHomeprazole) was notably different for
homozygote’s with the variant allele when compared with homozygote’s with the wild-type
allele (19).
Genotypes: CYP2C19*2/*2, CYP2C19*2/*3 which are poor metabolizes inactivates the
Omeprazole activity and shows intermediate activity with the genotypes CYP2C19*1/*2 and
have extensive activity towards CYP2C19*17*17, CYP2C19*1/*17, CYP2C19*2/*17 (20).
Clopidogrel is an antiplatelet prodrug that undergoes metabolic activation by CYP3A4 and
CYP2C19 (21). Previously reported meta-analyses have constantly documented that carriers
of CYP2C19*2 have an impaired antiplatelet effect in clopidogrel-treated patients, resulting
in higher risk of the recurrence of major adverse cardiovascular events(MACE), stent
thrombosis and even death (22-25). Several studies have evaluated the effects of the
CYP2C19*17 variant on adenosine diphosphate (ADP) - induced platelet aggregation and
clinical outcomes (e.g. MACE, stent thrombosis, bleeding or death) in patients treated with
Clopidogrel, which demonstrate that the presence of the CYP2C19*17 variant would increase
not only clinical efficacy, but also bleeding risk (26).
Tamoxifen is an anti-oestrogenic compound, commonly used in oestrogen receptor-positive
breast cancer. Previously published case control studies have shown that CYP2C19*17 was
surprisingly identified as a predictor of better tamoxifen response relative to carriers of alleles
9
(CYPC19*1, *2 and *3) associated with impaired enzyme activity (21). As CYP2C19 has
been shown to catalyze metabolism of oestrogen and testosterone, it has been reported to
affect the risk of oestrogen receptor  positive breast cancer in postmenopausal women.
Published articles shows that CYP2C19*2 is involved in increased level of oestrogen levels
with either heterozygousity or homozygousity while the extensive variant CYP2C19*17 was
associated with low oestrogen level and decreased breast cancer risk. Hence it has been
shown that CYP2C19*2 is associated with a favorable progression-free survival in patients
with metastatic breast cancer treated with tamoxifen, while carriers of CYP2C19*17 allele
who were treated with adjuvant tamoxifen had favorable disease-free survival compared to
non CYP2C19*17 carries (27-30).
Our methods may require further troubleshooting and optimization to get an optimal PCR
condition and should consider genotyping CYP2C19*3 variant along with this assay as it
would be useful for our routine laboratory use. The results of the single variant T-ARMS
multiplexed PCR can be obtained within 3hrs hence it can be implemented in the clinical lab.
As the genetic variants of CYP2C19*2 and CYP2C19*17 show a significant association with
the therapeutic dose of approximately 15% of all prescribed mainstream drugs, it is important
to genotype these variants among the patient population. By implementing this genotyping
assay, it would be a medically and economically beneficial pharmacogenomic platform which
can be used as an aid in determining personalized therapeutic strategies.
10
REFERENCE
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Tomalik-Scharte D, Lazar A, Fuhr U, Kirchheiner J. The clinical role of genetic
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De Morais SM, Wilkinson GR, Blaisdell J, Meyer UA, Nakamura K, Goldstein JA.
Identification of a new genetic defect responsible for the polymorphism of (S)mephenytoin metabolism in Japanese. Mol Pharmacol. 1994 Oct;46(4):594-8.
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Küpfer A, Preisig R. Pharmacogenetics of mephenytoin: A new drug hydroxylation
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Nakamura K, Goto F, Ray WA, McAllister CB, Jacqz E, Wilkinson GR, Branch RA.
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Bertilsson L. Geographical/Interracial Differences in Polymorphic Drug Oxidation. ClinPharmacokinet. 1995 1995/09/01;29(3):192-209.
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Desta Z, Zhao X, Shin J-G, Flockhart D. Clinical Significance of the Cytochrome P450
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de Morais SM, Wilkinson GR, Blaisdell J, Nakamura K, Meyer UA, Goldstein JA. The
major genetic defect responsible for the polymorphism of S-mephenytoin metabolism in
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Rosemary J, Adithan C. The pharmacogenetics of CYP2C9 and CYP2C19: ethnic
variation and clinical significance. Curr Clin Pharmacol. 2007 Jan;2(1):93-109.
9.
Frere C, Cuisset T, Morange PE, Quilici J, Camoin-Jau L, Saut N, Faille D, Lambert M,
Juhan-Vague I, Bonnet JL, Alessi MC. Effect of cytochrome p450 polymorphisms on
11
platelet reactivity after treatment with clopidogrel in acute coronary syndrome. Am J
Cardiol. 2008 Apr 15;101(8):1088-93.
10. Nyiro G, Inczedy-Farkas G, Remenyi V, Gal A, Pal Z, Molnar MJ. The effect of the
CYP 2C19*2 polymorphism on stroke care. Acta Physiol Hung. 2012 Mar;99(1):33-9.
11. Mizutani T. PM frequencies of major CYPs in Asians and Caucasians. Drug Metab Rev.
2003 May-Aug;35(2-3):99-106.
12. Cuisset T, Loosveld M, Morange PE, Quilici J, Moro PJ, Saut N, Gaborit B, Castelli C,
Beguin S, Grosdidier C, Fourcade L, Bonnet JL, Alessi MC. CYP2C19*2 and *17 alleles
have a significant impact on platelet response and bleeding risk in patients treated with
prasugrel
after
acute
coronary
syndrome.
JACC
Cardiovasc
Interv.
2012
Dec;5(12):1280-7.
13. Rychlik W, Spencer WJ, Rhoads RE. Optimization of the annealing temperature for
DNA amplification in vitro. Nucleic Acids Res. 1990 Nov 11;18(21):6409-12.
14. King CR, Porche-Sorbet RM, Gage BF, Ridker PM, Renaud Y, Phillips MS, Eby C.
Performance of commercial platforms for rapid genotyping of polymorphisms affecting
warfarin dose. Am J Clin Pathol. 2008 Jun;129(6):876-83.
15. Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH. Multiplex PCR: critical
parameters and step-by-step protocol. Biotechniques. 1997 Sep;23(3):504-11.
16. Hsiao SJ, Rai AJ. Multiplexed Pharmacogenetic Assays for SNP Genotyping: Tools and
Techniques for Individualizing Patient Therapy.
17. Ogu CC, Maxa JL. Drug interactions due to cytochrome P450. Proceedings (Baylor
University Medical Center). 2000;13(4):421.
18. Balian JD, Sukhova N, Harris JW, Hewett J, Pickle L, Goldstein JA, Woosley RL,
Flockhart DA. The hydroxylation of omeprazole correlates with S-mephenytoin
metabolism: a population study. Clin Pharmacol Ther. 1995 Jun;57(6):662-9.
12
19.
Sim SC, Risinger C, Dahl ML, Aklillu E, Christensen M, Bertilsson L, IngelmanSundberg M. A common novel CYP2C19 gene variant causes ultrarapid drug
metabolism relevant for the drug response to proton pump inhibitors and
antidepressants. Clin Pharmacol Ther. 2006 Jan;79(1):103-13.
20.
Chiba K, Shimizu K, Kato M, Nishibayashi T, Terada K, Izumo N, Sugiyama Y.
Prediction of inter-individual variability in pharmacokinetics of CYP2C19 substrates in
humans. Drug Metab Pharmacokinet. 2014 Apr 15.
21.
Li‐Wan‐Po A, Girard T, Farndon P, Cooley C, Lithgow J. Pharmacogenetics of
CYP2C19: functional and clinical implications of a new variant CYP2C19* 17. British
journal of clinical pharmacology. 2010;69(3):222-30.
22.
Hulot JS, Collet JP, Silvain J, Pena A, Bellemain-Appaix A, Barthelemy O, Cayla G,
Beygui F, Montalescot G. Cardiovascular risk in clopidogrel-treated patients according
to cytochrome P450 2C19*2 loss-of-function allele or proton pump inhibitor
coadministration: a systematic meta-analysis. J Am Coll Cardiol. 2010 Jul 6;56(2):13443.
23.
Jin B, Ni HC, Shen W, Li J, Shi HM, Li Y. Cytochrome P450 2C19 polymorphism is
associated with poor clinical outcomes in coronary artery disease patients treated with
clopidogrel. Mol Biol Rep. 2011 Mar;38(3):1697-702.
24.
Sofi F, Giusti B, Marcucci R, Gori AM, Abbate R, Gensini GF. Cytochrome P450
2C19*2 polymorphism and cardiovascular recurrences in patients taking clopidogrel: a
meta-analysis. Pharmacogenomics J. 2011 Jun;11(3):199-206.
25.
Zabalza M, Subirana I, Sala J, Lluis-Ganella C, Lucas G, Tomas M, Masia R, Marrugat
J, Brugada R, Elosua R. Meta-analyses of the association between cytochrome
CYP2C19 loss- and gain-of-function polymorphisms and cardiovascular outcomes in
13
patients with coronary artery disease treated with clopidogrel. Heart. 2012
Jan;98(2):100-8.
26.
Li Y, Tang HL, Hu YF, Xie HG. The gain-of-function variant allele CYP2C19*17: a
double-edged sword between thrombosis and bleeding in clopidogrel-treated patients. J
Thromb Haemost. 2012 Feb;10(2):199-206.
27.
Beelen K, Opdam M, Severson TM, Koornstra RH, Vincent AD, Hauptmann M, van
Schaik RH, Berns EM, Vermorken JB, van Diest PJ, Linn SC. CYP2C19 2 predicts
substantial tamoxifen benefit in postmenopausal breast cancer patients randomized
between adjuvant tamoxifen and no systemic treatment. Breast Cancer Res Treat. 2013
Jun;139(3):649-55.
28.
Justenhoven C, Hamann U, Pierl CB, Baisch C, Harth V, Rabstein S, Spickenheuer A,
Pesch B, Bruning T, Winter S, Ko YD, Brauch H. CYP2C19*17 is associated with
decreased breast cancer risk. Breast Cancer Res Treat. 2009 May;115(2):391-6.
29.
Ruiter R, Bijl MJ, van Schaik RHN, Berns EMJJ, Hofman A, Coebergh J-WW, van
Noord C, Visser LE, Stricker BH. CYP2C19*2 polymorphism is associated with
increased survival in breast cancer patients using tamoxifen. Pharmacogenomics. 2010
2010/10/01;11(10):1367-75.
30.
Li-Wan-Po A, Girard T, Farndon P, Cooley C, Lithgow J. Pharmacogenetics of
CYP2C19: functional and clinical implications of a new variant CYP2C19*17. British
journal of clinical pharmacology. 2010;69(3):222-30.
14
SOP
:
CYP2C19 Genotyping
Title
:
Detection of the CYP2C19 variant alleles
Last Revised
:
April 2014
Test
:
Molecular Genetic Test
Purpose
:
Detect the presence of CYP2C19*2 and *17 variants to predict the effect of drug
metabolism.
Method
:
Multiplex Allele- Specific PCR (AS-PCR)
1. DNA extraction
2. Multiplex allele specific PCR
3. Agarose gel electrophoresis
Step 1
Primer reconstitution (SOP003).
PRIMER
SEQUENCE
CYP2C19*2
CYP2C19*17
Step 1
:
F(G)
R(A)
C[F]
C[R]
F(T)
R(C)
C[F]
C[R]
5’ - ACT ATC ATT GAT TAT TTC CCG - 3’
5’ - GTA ATT TGT TAT GGG TTC CT -3’
5’ - CAG AGC TTG GCA TAT TGT ATC -3’
5’- TAT CGC AAG CAG TCA CATAAC -3’
5’ -TGT CTT CTG TTC TCA AAG TA -3’
5’- ATTA TCT CTT ACA TCA GAG ATG-3’
5’- AAGAAG CCT TAG TTT CTC AAG -3’
5’- AAACACCTTTACCATTTAACCC -3’
DNA extraction using peripheral blood leucocytes (Ref.SOP001/SOP002)
1
Step 2
:
Set-up PCR:25µL Master mix contains;
19.
20.
21.
22.
23.
Master Mix
ddH2O
5x buffer
MgCl2(25mM)
dNTPmix(10mM)
CYP2C19*2 F(C)
CYP2C19*2 R(T)
CYP2C19*2 C[F]
CYP2C19*2 C[R]
CYP2C19*17 F(T)
2
12.5 µL
5.0 µL
1.5 µL
1.0 µL
0.6 µL
24. CYP2C19*17 R(C)
0.6 µL
25. CYP2C19*17 C[F]
1.0µL
26. CYP2C19*17 C[R]
0.6µL
Taq
DNA
Total
:
Step 3
Step 4
1
10.3 µL
5.0 µL
1.5 µL
1.0 µL
1.0 µL
1.0 µL
1.5µL
1.5µL
:
0.2
2.0
25µL
0.2
2.0
25µL
Run PCR
Initial denaturation at 95˚c for 10 mins.
Denaturation at 95 ˚c for 30 sec.
Annealing at 53 ˚c for 30 sec.
5 cycles
Extension at 72 ˚c for 30 sec.
Denaturation at 95 ˚c for 30 sec.
Annealing at 57 ˚c for 30 sec.
15 cycles
Extension at 72 ˚c for 30 sec
Denaturation at 95 ˚c for 30 sec.
Annealing at 60 ˚c for 30 sec.
15 cycles
Extension at 72 ˚c for 30 sec
Final extension at 72 ˚c for 7 min.
Cooling at 4 ˚c ∞
35 Cycles
Analysis by gel electrophoresis (Ref:SOP004)
Run 10µl of PCR product in a 3% gel at 80v in for 30 min.
2
Gel
:
Interpretation
Allele
CYP2c19*2 Outer
Band sizes
291bp
CYP2C19*2
F(G)wild
CYP2C19*2 R(A)
CYP2C19*17 Outer
201bp
CYP2C19*17 F(T)
CYP2C19*17
R(C)wild
216bp
294bp
128bp
506bp
:
Reference
1.
2.
Cuisset, T., et al., CYP2C19*2 and *17 alleles have a significant impact on
platelet response and bleeding risk in patients treated with prasugrel after acute
coronary syndrome. JACC Cardiovasc Interv, 2012. 5(12): p. 1280-7.
PCR conditions modified. Band sizes were reevaluated.
3
Cytochrome P450 2C19*2 and 2C19*17 Genotyping
Human Genetics Unit, Faculty of Medicine, University of Colombo
The cytochrome P450 2C19 (CYP2C19) is
important for the metabolism of several
therapeutic agents such as proton pump
inhibitors (PPIs), anticonvulsants, antiulcer, anti
seizure and antidepressants drugs such as
Omeprazole, Tamoxifen, Clopidogrel fluoxetine,
lansoprazole,
S-mephenytoin,
tolbutamide,
voriconazole, diazepam. Large inter-individual
differences have been seen in the metabolism of
these drugs in vivo, and individuals can be divided
in to normal (extensive metabolizer, EM),
intermediate metabolizer (IM), poor metabolizer
(PMs) and ultra rapid metabolizers (UM).
Genetic polymorphism CYP2C19*2 CYP2C19*17
exists, in 15–20% of Asian populations being poor
metabolizers with no CYP2C19 function in which
may reduce the efficacy of clopidogrel.
CYP2C19*2(rs4244285), the most common variant
allele of CYP2C19 is associated with a distinct
decrease in platelet response to clopidogrel in
unsteady patients and an impaired prediction in
clopidogrel-treated
patients.
CYP2C19*17
(rs12248560) is a recently identified variant in
CYP2C19 gene, carriers of this variant were found
to have a significantly lower AUC of Omeprazole,
suggesting that this would give rise to an
extensive metabolizer phenotype.
Reasons for Referral:
Evaluating the commonly seen genetic factors in Asians
and Caucasians which affecting the drug metabolism for
patients taking drugs such as Clopidogrel (Plavix), Antidepressants such as Amitriptyline and Escitalopram,
Tamoxifen and Omeprazole.
Limitations:

Only the most common two alleles are targeted by
this assay, due to the lower prevalence of the other
alleles in the country, they are not tested.

Non genetic factors and variants in other genes
involving the drug metabolism are not measured by this
assay.

Results of this test should be interpreted in the
context of clinical presentation and inconsultation with a
licensed geneticist and/or pharmacist.
Testing Methodology:
This test is performed by DNA amplification
using a tetra primer Amplification-refractory
mutation system polymerase chain reaction (TARMS PCR) followed by gel electrophoresis.
Variant Alleles Tested:
*2 (c. 681G>A) - Reduced activity
*17(c.-806C>T) - Increased activity
Specimen Requirements:



Peripheral blood: Collected in EDTA (purple
top) tube.
Adults: 3cc
Handling: Room Temp- specimen processed
within 72 hours
Turnaround Time:
14 days
Perforating Laboratory:
Human Genetics Unit
Faculty of Medicine
University of Colombo
Kynsey Road, Colombo 8, Sri Lanka
Phone (94-011) 2695 300, 2689 545 (Direct)
Fax (94-011) 2689979
[email protected]
http://www.hgucolombo.org
References:
Tomalik-Scharte D, Lazar A, Fuhr U, Kirchheiner J. The
clinical role of genetic polymorphisms in drugmetabolizing enzymes. Pharmacogenomics J. 2008
Feb;8(1):4-15.
De Morais SM, Wilkinson GR, Blaisdell J, Meyer UA,
Nakamura K, Goldstein JA. Identification of a new
genetic defect responsible for the polymorphism of (S)mephenytoin metabolism in Japanese. Mol Pharmacol.
1994
Oct;46(4):594-8.
Human Genetics Unit
Faculty of Medicine
University of Colombo
Kynsey Road, Colombo 8, Sri Lanka
Phone (94-011) 2695 300, 2689 545 (Direct)
Fax (94-011) 2689979
[email protected]
http://www.hgucolombo.org
Confidential Molecular Genetic Laboratory Test Report
Patient Identification:
Name:
Date :
Age:
Sex:
Lab Reference:
Indication:
Material Tested:
EDTA Blood
Test:
CYP2C19*2 and *17 genotyping
Analysis Performed:
The following mutations were genotyped by T-ARMS PCR:
CYP2C19*2 ( rs424428,, 681G>A )
CYP2C19*17 ( rs12248560, C>T)
Result:
NAME OF GENE: CYP2C19
LOCATION OF GENE: 10q24.1
Variants tested
RESULT
rs1799853 (CYP2C19*2)
GG
rs1057910 (CYP2C19*17)
CC
Poor/ Intermediate/ ultra rapid metabolizes.
Remaks:
Risk interpretation based on Coriell's CYP2C19 Clopidogrel Metabolizer
Type Genotype Translation Version 1 (March 2011)
Test Limitations: There may be other variants in the CYP2C19 gene that are
not included in this test, that influence metabolizer state.
Prof. Vajira H. W. Dissanayake MBBS, PhD
Medical Geneticist
Analysis Performed by:…………………………
Analysis Requested by:
Because of their complexity and their potential implications for other family members, all genetic tests should be
accompanied by genetic counseling.
Prof. Rohan W. Jayasekara MBBS (Ceylon), PhD (Newcastle), C.Biol., MSB (London) – Medical Geneticist and Director
Prof. Vajira H. W. Dissanayake MBBS (Colombo), PhD (Nottingham) – Medical Geneticist
MOLECULAR CYTOGENETICS
REPORT
MOLECULAR CYTOGENETIC
CHARACTERIZATION OF THE FIRST
REPORTED SRI LANKAN CHILD WITH
A DE NOVO 9p INVERTED
DUPLICATION (p13.3;p23)
ABSTRACT
In this case report we describe a child with a de novo inverted duplication in the (p13.3;p23)
region of chromosome 9. The child presented with dysmorphic features developmental delay,
craniofacial abnormalities such as bulbous nose, hypertelorism, large bat ear, limb
abnormalities such as fingers and toes with small nails, fifth-finger clinodactyly. To our
knowledge this is the first reported case of a child with de novo 9p inverted duplication in Sri
Lanka. Molecular cytogenetic characterization confirmed that the phenotypic features
observed in this child is in concordance to the spectrum of clinical features seen in children
with duplication in the proximal 9p region which involves the duplication of the 9p22.3-p23
critical region.
KEY WORDS: Inverted, duplication, (9)(p13.3;p23), developmental delay, de novo,
(9p22.3;p23), FISH
1
INTRODUCTION
Compared to other rare chromosomal disorders, duplication of the short arm of chromosome
9 (partial trisomy9p) is not uncommon. The first reported case was in 1970 (1) and more than
150 patients have been reported so far. Trisomy 9p is the fourth most common chromosome
anomaly in a live-born after abnormalities in chromosomes 21, 18, and 13 (2). In majority of
patients, the trisomic segment of reported 9p duplications was transmitted from a parent
carrying a reciprocal balanced translocation and only a small number arose from de novo
duplications (3). De novo duplications of this chromosomal region have been previously
described in approximately 15 patients up to date (4, 5). Patients with partial trisomy of the
short arm of chromosome 9 often present a wide spectrum of phenotype including
developmental delay, craniofacial abnormalities such as bulbous nose, hypertelorism and
limb abnormalities such as fingers and toes with small nails and fifth-finger clinodactyly.
This paper describes the first reported Sri Lankan child with a de novo 9p inverted
duplication, detected by conventional cytogenetic analysis, characterized and delineated
further by Fluorescent in situ Hybridization (FISH).
CASE PRESENTATION
Here we present a four year old boy born after an uneventful pregnancy as the first child to
healthy non consanguineous parents. His weight, length and occipital frontal circumference
(OFC) at present were 12kg (just below 3rd centile), 94 cm (just below 3rd centile) and 48cm
respectively. The mother had been on antibiotics for a urinary tract infection during the first
trimester in the antenatal period. An ultrasound scan of the brain showed mild dilation of the
ventricular system and a computed tomography (CT) scan showed mild dilated lateral
ventricles. Testosterone, Follicle-stimulating hormone and luteinizing hormone (FSH/LH)
levels and Thyroid-stimulating hormone (T4/TSH) levels were in the normal range.
2
The following dysmorphic features were noted: strabismus, low set anteverted large ears (bat
ears), broad nasal root, short philtres, a mouth with downturned corners with a prominent
lower lip, short and broad hands with short fingers with 5th finger clindodactyly, bilateral
simian creases and micropenis with bilateral testis (Figure 1). He presented delayed bone age,
developmental delay, speech delay and minor intellectual disability .The child has undergone
X-ray and Ultra sound scans to exclude internal pathologies and limb dysmorphisms. Fragile
X syndrome was excluded by trinucleotide repeat expansions test for FMR 1 gene
and
mucopolysaccharidosis was excluded by berry spot test.
Figure 1. Clinical phenotype of the patient
(A) Side view showing frontal bossing (B) front view showing low set anteverted large ears
(bat ears), broad nasal root, short philtres, a mouth with downturned corners with a prominent
lower lip, (C) short and broad hands with short fingers with 5th finger clindodactyly and
bilateral simian creases (D) short leg showing sandal gap (E) micropenis with bilateral testis
3
MATERIALS AND METHODS
Ethical Clearance
The ethical approval was obtained from the ethic review committee, Faculty of Medicine,
University of Colombo. The parents gave the written informed consent for further genetic
evaluation, photography and publication.
Conventional karyotyping and Fluorescence in situ Hybridization (FISH)
Metaphase chromosome spreads preparation from the patient and his parents’ peripheral
blood lymphocyte cultures and GTL-banding were performed according to standard methods.
Twenty metaphase spreads were captured by using Olympus BX61 epifluorescence
microscope (Olympus, Tokyo, Japan) and analysed using CytoVision® software (Applied
Imaging, Dornach, Germany). The maximum band resolution achieved in the proband’s
spreads was 550 bands according to the International System of Cytogenetic Nomenclature
2009. (Figure 2)
FISH was performed on the metaphase chromosome spreads of the patient using gene/region
specific FISH probes (Empire Genomics, Roswell Park Cancer Institute in Buffalo, New
York, USA) according to the manufacture’s protocol. Initially, the cell culture was harvested
using standard Cytogenetic protocol. The fixative (Carnoy’s 3:1 methanol: acetic acid) in
sample tube was changed until supernatant is colourless then refixed in fresh fixative prior to
slide preparation. The slides were cleaned by placing in a coplin jar with 70% alcohol for 5
minutes, wiping vigorously in one direction several times with a tissue to remove debris, then
placing in a coplin jar with fresh fixative. Three drops of suspension were added to the slide
in a vertical angle. Then the slides were gently rotated, to make a thin cell suspension and
were kept parallel until a grainy appearance was observed and to drain the excess suspension.
4
The BAC clones used for the specific genes were RP11-627M21 for DMRT 1 gene located at
9p24.3 and RP11-145E14 for RMRP gene located at 9p13.3 (BAC clone database, Empire
Genomics, Roswell Park Cancer Institute in Buffalo, New York, USA). The probes were
fluorescently labeled from the time of manufacture with spectrum Green and spectrum Red
respectively (Figure 3). From each of the probe mixtures, 10μl were added on to the slides
(2ul probe + 8ul hybridization buffer) separately. A clean 22 x 22 mm cover slip was applied
on to each slide and the edges were sealed using rubber cement. The probes were hybridized
with metaphase chromosomes in a StatSpin®ThermoBrite® Hybridizer (Abbot molecular);
denaturation at 73°C for 2 minutes/hybridize at 37°C for 16 hours. After the hybridization
the slides were taken and the cover slips were removed and were placed in a pre-warmed
WS1 (0.4xSSC/0.3% NP-40) at 73°C solution and let stand in WS1 (agitating ~10sec)
exactly 2min. Then the slides were transferred to WS2 (2xSSC/0.1% NP-40) at room
temp/1min. The slides were dried in the dark, and counterstained with 10μl of 4’, 6diamidino-2-phenylindole (DAPI) and covered with 22 x 22 mm cover slips. After 15-30
minutes the slides were visualized and the images were captured using Olympus BX61
epifluorescence microscope (Olympus, Tokyo, Japan), ×100/1.3 magnification objective with
CCD camera model ER-3339 (Applied Imaging, Newcastle, UK) and analyzed using
GenASIs software (Applied Spectral Imaging, USA).
5
RESULTS
Cytogenetic analysis detected a karyotype of 46,XY,add(9pter). The parents’ karyotypes
were normal therefore this was a de novo rearrangement.
According to the banding pattern of the patient, we suspected the abnormality was a 9p
duplication hence FISH analysis was performed to confirm the diagnosis. In FISH analysis,
the probe targeting the RMRP gene at 9p13.3 which was used to confirm the duplication
showed 4 signals (spectrum red) indicating that the patient had an inverted duplication 9p.
The probe targeting the DMRT 1 gene at 9p24.3 terminal which was used to verify a terminal
deletion/duplication showed 2 signals (spectrum green) indicating the presence of one DMRT
1 gene in the derivative chromosome (Figure 3). Hence the final karyotype of the patient after
molecular-cytogenetic characterization was 46,XY,dup(9)(p13.3;p23) (Figure 2).
Figure 2. Identification of the 9p inverted duplication by conventional karyotyping
Ideogram of chromosome 9 showing the normal and the inverted duplicated region (red,
green, red markers) and cut-out of the abnormal and normal chromosome 9 in G-banding at a
resolution of 550 bands.
6
Figure 3. Characterization of the 9p inverted duplication by FISH analysis.
(A) and (B) FISH analysis using spectrum red for RMRP gene at 9p13.3, (C) and (D) FISH
analysis using spectrum green for DMRT 1 gene at 9p24.3. Arrow indicates the inverted
duplication of 9p13.3 region.
DISCUSSION
Partial trisomy 9p/duplication 9p is one of the most common autosomal structural anomalies.
The phenotype–genotype correlation of this rearrangement has been well described in the
scientific literature with more than 150 patients reported. Most of these reported duplications
are due to malsegregation of chromosomes inherited from a parent with a chromosome 9
reciprocal translocation, with fairly a minority of pure de novo 9p duplications (3, 6). With
regards to the variation in size of the 9p duplications, this syndrome is characterized by
7
typical
dysmorphic
features:
growth
and
intellectual/
developmental
delay,
microbrachcephaly, deep and wide-set eyes with down slanting palpebral fissures, prominent
nasal root with a bulbous nasal tip, downturned corners of the mouth, lowset ears, short
fingers and toes with hypoplastic nails, and delayed bone age.
There are reports of children with different duplications between 9p11.2 and 9p22.1 with
normal development. Some had few facial and upper limb abnormalities associated with
trisomy 9p, while the others had insignificant features. Duplications of 9p11.2 to 9p13.1 are
believed to be a natural chromosome variant with no reported abnormal phenotypes. The
9p22.1;p23 region had been proposed to be the critical region for the 9p duplication
syndrome phenotype. (7, 8) Hence, the association of region 9p22 to p23 in our patient
corresponds with the general phenotypic features of the 9p duplication syndrome.
In previous publications, a large partial trisomy 9p12;p21.3 with normal phenotype (9) has
been reported. There were publications of a 16.6Mb interstitial duplication of the
9p13.2;p21.3 segment in a patient with dysmorphic features similar to that of the 9p
duplication syndrome with speech and language delays but with normal mentality (10) and a
20Mb 9p13.1;p22.1 duplication in a girl with mild dysmorphic features and low weight
increase but a normal Intelligence quotient (IQ) (7). It has been postulated that the critical
region of speech delay/ langue delay in the 9p duplication syndrome could be the 4.9Mb of
the 9p21.2;p21.3 region (10). Summarized phenotypic manifestations of our patient
compared with the documented 9p-duplication syndrome and other reported 9p13p21-22
duplication is shown in Table 1. Most of the clinical features; Speech/langue delay,
macrocephaly, down-slanting palpebral fissures, hypertelorism, bulbous/prominent nose,
down-turned mouth, low-set ears/abnormal large ears, single palmar crease, clinodactyly,
delayed bone age are aligning with the previously reported 9p duplication cases.
8
Published reports show that haploinsufficiency of DMRT 1, 2, and 3 result in gonadal
abnormalities in male (11). Though in our patient, the expected FISH singles for DMRT 1
gene were seen, the inversion of the duplicated segment which has involved the 9p terminal
region must have disrupted one of the above mentioned genes in order to give the
phenotypes; micropenis. The commonly seen phenotype of RMRP gene duplication ranging
from milde to severe hypodysplasia was not seen in our patient.
To our knowledge this is the first case reported of a child with de novo 9p inverted
duplication in Sri Lanka. In conclusion, molecular cytogenetic characterization confirmed
that the phenotypic features observed in this child is in concordance to the spectrum of
clinical features seen in children with duplication in the proximal 9p region which involves
the duplication of the 9p22.3-p23 critical region.
9
Table 1. Phonotypical Findings in Five Patients with Pure dup(9)(p13p21–22)
Major features seen in
duplication 9p syndrome
Duplication 9p
Mental retardation
Speech/langue delay
Short stature
Micro/brachy/dolichocephaly
Down-slanting palpebral
fissures
Hypertelorism
Bulbous/prominent nose
Down-turned mouth
Low-set ears/abnormal large
ears
Single palmar crease
Clinodactyly/brachydactyly
Dysplasia or hypoplasia of
nails
Delayed bone age
A(12)
B(13)
C(9)
D(7)
E(10)
F(14)
p13p22.1,
Direct
+
P13.2p21.3,
inverted
+
+
-
p13-p24,
inverted
+
+
Mild
+
-
-
-
-
-
+
Mild
+
+
+
+
+
+
+
+
+
+
+
+
+
p13-p22,
tandem
p13p21
p13p21.3
Mild
+
-
+
+
+
+
+
+
+
+
+
Mild
+
+
+
+
+
+
+
Present
case
p13.3-p23,
inverted
+
+ present; - absent; blank, not mentioned in report.
10
REFERENCES
1.
Rethore MO, Larget-Piet L, Abonyi D, Boeswillwald M, Berger R, Carpentier S,
Cruveiller J, Dutrillau B, Lafourcade J, Penneau M, Lejeune J. [4 cases of trisomy for the
short arm of chromosome 9. Individualization of a new morbid entity]. Ann Genet. 1970
Dec;13(4):217-32.
2.
Al Achkar W, Wafa A, Moassass F, Liehr T. Partial trisomy 9p22 to 9p24.2 in
combination with partial monosomy 9pter in a Syrian girl. Mol Cytogenet. 2010;3:18.
3.
Hulick PJ, Noonan KM, Kulkarni S, Donovan DJ, Listewnik M, Ihm C, Stoler JM,
Weremowicz S. Cytogenetic and array-CGH characterization of a complex de novo
rearrangement involving duplication and deletion of 9p and clinical findings in a 4month-old female. Cytogenet Genome Res. 2009;126(3):305-12.
4.
Tsezou A, Kitsiou S, Galla A, Petersen MB, Karadima G, Syrrou M, Sahlen S, Blennow
E. Molecular cytogenetic characterization and origin of two de novo duplication 9p
cases. Am J Med Genet. 2000 Mar 13;91(2):102-6.
5.
Fujimoto A, Lin MS, Schwartz S. Direct duplication of 9p22-->p24 in a child with
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