Pathology – Research and Practice 207 (2011) 671–673

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Pathology – Research and Practice

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Original article

Thrombosis risk in carriers of the factor V Leiden mutation: Is it associated with a

defined skin color?
Ali A. Dashti a,∗, Mehrez M. Jadaon a, Abdulsamad M. Abdulsamad b, Mohammad H. Dashti c, Hend L. Lewis a

a
Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, Kuwait University, Kuwait
b
Department of Surgery, Al-Amiri Hospital, Kuwait
c
Department of Surgery, Mubarak Al-Kabeer Hospital, Kuwait

a r t i c l e i n f o a b s t r a c t

Article history: Factor V Leiden (FVL; G1691A) is an autosomal dominant mutation with a high risk for thrombosis.
Received 30 January 2011 Speculation that founders of FVL lived in the Middle East is supported by a prevalence of FVL that is
Received in revised form 4 June 2011 higher in Arabs residing in Israel, Jordan, Lebanon, and Syria (12–14%) than in other white populations
Accepted 27 July 2011
like Europeans (4–5%, up to 15% in the South of Sweden). We sought to verify the appropriate use of
skin color as a clinical sign by which Arab individuals in Kuwait are included or excluded from testing
Keywords:
for FVL. After institutional approval, 200 healthy Arabs residing in Kuwait consented to participate. Skin
Kuwait
type was distinguished for the participants by Fitzpatrick natural skin color classification: 76 (38%) skin
Factor V Leiden mutation
Arab
type II (white), 96 (48%) Mediterranean skin type IV (brown), and 28 (14%) skin type VI (black). FVL was
Skin color tested by real-time PCR, and the percentage of carriers was calculated in each group. FVL was positive
in 17 (8.5%) of the total subjects: 8 (10.5%) skin type II, 7 (7.3%) skin type IV, and 2 (7.1%) skin type VI.
Therefore, FVL shows an even distribution in Arabs, and all Arabs residing in Kuwait should be tested for
FVL irrespective of skin color.
© 2011 Elsevier GmbH. All rights reserved.

Introduction
Factor V Leiden (FVL) is a point mutation from arginine
to glutamine in the factor V amino acid residue number 506
(Arg506Gln). Factor V is a regulatory blood clotting factor respon-
sible for maintaining hemostasis [7,9,16]. FVL is the most common
monogenic disorder whose origin and geographic distribution
within populations with white skin has received much attention
[4,6,9,12,13,19,25,26,28,35]. The consensus is that it is nonexistent
in the native populations of Africa, America, Asia, and Australia
[1,6,13,25] with numerous reports attributing white skin to the
founders of both the prothrombin 20210G > A mutation and FVL
[9,36]. Cox et al. estimate the FVL as a single ancient mutation which
occurred in white European founding population within the last
100,000 years [9]. Zivelin et al. hypothesize a single origin that prob-
ably occurred after the divergence of Africans from non Africans
and of Caucasoid from Mongolian subpopulations, around 32,000
years ago [38], while the single origin is estimated by others to
have occurred in whites approximately 24,000 years ago toward
the end of the last Pleistocene glaciations [37]. FVL has also been
identified in an Urartian dating back to 1000 BC [1]. Although FVL
is reported as almost absent in non-European whites, there is a
high prevalence of the FVL in the Middle East, and the possibility
that the founders lived around this region is supported by the find-
ing of a single haplotype for the mutation in Mediterranean and
Indian individuals [8]. We and others have reported a high inci-
dence of FVL in Arab individuals with venous thromboembolism
(VTE) and in healthy controls living in Kuwait, Lebanon, Palestine,
Egypt, and Syria [2,3,5,10,11,14–16,17,20,22,24,29–32]. Skin pig-
mentation in Arabs according to the Fitzpatrick classification varies
from skin type II (white) to Mediterranean skin type IV (brown) or
skin type VI (black). Dr. Fitzpatrick was a Harvard dermatologist
who classified the person’s skin color according to their tolerance of
sunlight [18]. This study seeks to verify the appropriate use of skin
color as a clinical sign by which individuals in Kuwait are included
or excluded from testing for FVL. The majority of Arabs residing
in Kuwait are ethnic Kuwaitis, and others are from Syria, Jordan,
Palestine, Lebanon, and Egypt.

Materials and methods

∗ Corresponding author at: Department of Medical Laboratory Sciences, Faculty

Subjects, blood samples, and DNA extraction
of Allied Health Sciences – Kuwait University, P.O. Box 31470 – Sulaibekhat 90805
– Kuwait. Tel.: +965 66090302; fax: +965 24983835. Two hundred healthy Arab volunteers consented to provide
E-mail addresses: aad@hsc.edu.kw, dashti110@hotmail.com (A.A. Dashti). a venous blood sample (in EDTA anticoagulant) using standard

0344-0338/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.prp.2011.07.013
672 A.A. Dashti et al. / Pathology – Research and Practice 207 (2011) 671–673
Table 1
Number and percentage of cases with FVL in the three groups of Arabs categorized according to skin color: white (type II), brown (type IV), or black (type VI). Results were
either presence of the normal (G) allele on both chromosomes (normal wild-type GG), presence of the mutant FVL (A) allele on both chromosomes (homozygous AA), or
presence of one normal (G) allele on one chromosome and one mutant FVL (A) allele on the other chromosome (heterozygous GA). Allelic frequencies are also shown.
Skin color No. Wild-type GG FVL GA heterozygous
White (type II) 76 68 (89.5%) 7 (9.2%)
Brown (type IV) 96 89 (92.7%) 7 (7.3%)
Black (type VI) 28 26 (92.9%) 2 (7.1%)
Total 200 183 (91.5%) 16 (8.0%)
phlebotomy. The color of the skin of each volunteer was recorded
as white (type II), brown (type IV), or black (type VI) based on the
Fitzpatrick natural skin color classification [18]. Half of the sub-
jects were from Kuwait and the rest from Syria, Jordan, Palestine,
and Egypt. The principles of the Declaration of Helsinki were fol-
lowed, and the research was approved by the Ethical Committee of
the Health Sciences Center, Kuwait University, Kuwait.
Blood samples were used for DNA extraction either immedi-
ately or were stored at −20 ◦ C until being processed. A commercial
kit (JETQUICK for whole blood from Genomed company, Löhne,
Germany) was utilized for DNA extraction according to the man-
ufacturer’s instructions, and DNA samples were frozen at −20 ◦ C
until the next step. All volunteers gave a signed consent.
Real-time PCR
Each DNA sample was tested for FVL using specially designed
real-time PCR (rt-PCR) kit (FACTOR V Q – PCR Alert Kit from
Nanogen Advanced Diagnostics S. R. L., Buttigliera Alta, Italy). The
procedure was similar to a previous publication by the authors [11].
In brief, 5 ␮l of each DNA sample was mixed with 20 ␮l of a mixture
of primers and probes. The primers were designed by the company
to flank the region of factor V gene that contains the nucleotide
position 1691 where FVL is expected. The probes used were made to
specifically match either the wild-type G1691 or FVL mutant A1691
alleles. The wild-type and mutant probes were labeled with FAM
and VIC fluorophores, respectively, and both probes were blocked
by the MGB-NFQ group. The rt-PCR mixture was inoculated into
wells of a 96-well PCR reaction plate, and the rt-PCR reaction was
carried out in an AB 7500 FAST Real Time PCR System (Advanced
Biotechnologies, Foster City, CA, USA). The rt-PCR conditions were:
(1) Pre-Read run at 60 ◦ C for 2 min (to record the background sig-
nal); (2) Amplification run consisting of 3 steps: one hold at 50 ◦ C
for 2 min (UNG inactivation), one hold at 95 ◦ C for 10 min, and 40
cycles of alternating temperatures at 95 ◦ C for 15 s (denaturation)
and 60 ◦ C for 1 min (annealing and extension); (3) Post-Read run
at 60 ◦ C for 2 min (to subtract amplified signals from background
signal). For each case tested, the result was inferred as wild-type
alleles only (normal), mutant alleles only (homozygous FVL), or
both alleles (heterozygous FVL).
Statistical analysis
We studied the distribution of FVL as it relates to the three types
of skin pigmentation present in Arabs residing in Kuwait, namely
skin type II (white), Mediterranean skin type IV (brown), or skin
type VI (black). The percentage of FVL carriers was calculated to
the total number of volunteers in each group. These percentages
were compared to each other using Student’s t-test, considering a p-
value less than 0.05 as statistical significance. EpiCalc 2000 software
version 1.02 (downloaded from Joe Gilman and Mark Myatt, Brixton
books, at http://www.brixtonhealth.com/epicalc.html) was used to
perform the statistics.
Allelic frequencies were calculated for each group using the fol-
lowing formula:Allelic frequency = (heterozygous+2×homozygous)
2×total number of volunteers
FVL AA homozygous FVL heterozygous + homozygous Allelic frequency
1 (1.3%) 8 (10.5%) 0.0592
0 (0%) 7 (7.3%) 0.0365
0 (0%) 2 (7.1%) 0.0357
1 (0.5%) 17 (8.5%) 0.0450
Table 2
Results of Student’s t-test (p-values) comparing the percentages of FVL among the
three groups according to skin color. Any p-value ≤ 0.05 was considered statistically
significant.
White (type II) Brown (type IV) Black (type VI)
White (type II) – 0.318 0.443
Brown (type IV) 0.318 – 0.349
Black (type VI) 0.443 0.349 –
Results
Table 1 presents the numbers and percentages of FVL among the
three skin types. Among the 200 volunteers, 76 (38%) had skin type
II, 96 (48%) had skin type IV, and 28 (14%) had skin type VI. FVL was
positive in 8 of skin type II (10.5%), 7 of skin type IV (7.3%), and 2 of
skin type VI (7.1%). In total, 17 individuals had the mutation (8.5%),
16 of which were heterozygous and only one was homozygous (skin
type II volunteer). The general allelic frequency was 0.045. Table 2
presents statistical analysis results. There were no significant differ-
ences in the percentages of FVL among the three groups (p-values
were 0.318–0.443 which were >0.05).
Discussion
A genetic marker is a DNA sequence or a single nucleotide poly-
morphism with a known location on a chromosome that can be
used to identify a population. In that respect, the factor V Leiden, a
monogenic marker, identifies in individuals that carry the polymor-
phism a higher risk for developing thrombosis than in the general
population. It has been speculated that the founders of the muta-
tion lived in the Middle East because currently the prevalence of
this polymorphism is higher in Middle Eastern populations than in
other white populations [29,31]. Since Arab skin type varies from
white to brown and black, we sought to verify the appropriate use of
skin color as a clinical sign by which Arab individuals in Kuwait are
included or excluded from testing for FVL and to verify the assump-
tion that human skin color as a phenotypic trait has been incorrectly
used for centuries for the purpose of racial classification. Skin color
is a polygenic trait, and the genetic basis underlying normal varia-
tions in skin pigmentation, hair and eye color has been the subject of
intense research directed at understanding the diversity seen both
between and within human populations [27,34]. It is astonishing
that the understanding of molecular genetics of human pigmenta-
tion has progressed only recently with the discovery of important
pigment determining genes [21,23,33]. Skin color is profoundly
influenced by both the Kit ligand gene (KITLG), which produces
a cytokine that binds a receptor tyrosine kinase expressed on mast
cells, melanocytes, and germ cells, and the Agouti signaling peptide
(ASIP) that acts as an inverse agonist inhibiting eumelanin produc-
tion. KITLG is involved in the permanent survival, proliferation, and
migration of melanocytes. A KITLG mutation A326G (rs642742)
positively associated with variations in skin color occurs in over
80% of European and Asian and in less than 10% of Africans. Alle-
les in the vicinity of the ASIP are associated with skin color. The
ASIP inhibits eumelanin production by binding alpha-MSH, with a
frequency estimated roughly at 80% in Europeans, 75% in Asians,
 A.A. Dashti et al. / Pathology – Research and Practice 207 (2011) 671–673
and 20–25% in Africans. Thus, human white skin color cannot be
applied to biological race as skin color variation is clinal and not
well described by discrete racial categories. Clinal variation is a
common pattern of geographic variation that refers to a gradual
change in some feature across geography; for example the popu-
lation of mammals is smaller in the south and gradually become
larger and larger as one heads north in the northern hemisphere
[39].
Our article provides evidence that “white populations” of the
Middle East should include a wide range of skin color. Typical of
traits that show extensive natural selection, skin color shows a high
degree of variation specifically among Arabs and in this article we
demonstrate that it is not appropriate to use skin color in Kuwait
or elsewhere as a clinical sign by which individuals with hyper pig-
mented skin (dark color skin) are excluded from testing for the FVL
genetic polymorphism. Color of skin is a political, racial, ethnic,
societal, and cultural classification, and it should not be used as a
metaphor for race, at least in the clinical practice when FVL is the
issue.
Acknowledgement
The authors are grateful to Research Core Facility of the Health
Sciences Center, Kuwait University, for their assistance in the tech-
nical work (project numbers GM01/01 and GM01/05).
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