bir genetik araştırma
DISCUSSION
Which was the Initial Haplotype HbO-Arab Evolved on?
Approximately half of the normal Pomak chromosomes
display the patterns of Universal haplotypes I, II
and III while the other half displays two quite different
patterns, the rare Greek haplotype VI and the novel Pomak
haplotype I (Table 2).
The above-mentioned haplotypes are characteristic for
the Pomak population and have never been encountered
in Europe, with one unique exception concerning the
Greek haplotype VI which has been referred once in nor-
mal Greek chromosomes39. The 5´ subhaplotype pattern
of the latter is scarce in West Africa (2.9%), South Africa
(1.1%), Micronesia (0.5%) and Polynesia (0.4%). Haplotype
Pomak I, which is novel for Europe, encounters by
its 5´ subhaplotype in Melanesia (2.7%), Micronesia
(1.5%), Polynesia (0.7%) and Thailand (0.4%)17.
Grouping separately the respective 5´ and 3´ subhaplotypes
of the Greek haplotype VI and Pomak haplotype
I reveals more striking differences. Here, the 5´ subhaplotype
patterns 1(0)0(0)00(1)1 and 0(1)1(0)1(1)1 concentrate
all ambiguities, while the 3´ subhaplotype pattern
(?)1(?001)1 (? meaning: not examined) is not informative
as may represent the 3´ part of haplotypes I, II
or IX.
All 21 HbO-Arab carrying chromosomes are linked
with the common 3´ subhaplotype pattern (0)1(1001)1,
which is shared, among others, between Universal haplotype
I and Greek haplotype VI in the Pomak population.
On the other hand, only 3 out of the 21 HbO-Arab carrying
chromosomes displayed a common 5´ subhaplotype
pattern (found in Universal haplotype I) while the remaining
18 belonged uniformly to the 5´ subhaplotype pattern
of Greek haplotype VI. This implies that the HbO-Arab
mutation occurred on a pre-existing Greek haplotype VI
(type VIa) and its frequency increased considerably thereafter.
The hypothesis that HbO-Arab emerged on a preexisting
Greek haplotype VI and not vice-versa is supported
by the fact that the overall frequency of the Greek
VI 5´ subhaplotype among the normal genes of Pomak
origin (0.126) is six-fold higher in comparison with HbOArab
genes (0.021). This can be derived from the mean
HbO-Arab allele frequency in Pomaks (0.0273)4 and the
mean Greek VI 5´-subhaplotype frequency in normal
(0.129) and HbO-Arab Pomak chromosomes (0.857) (Table
2).
The recombination of the Greek haplotype VI 5´ subhaplotype
with a conventional 3´ subhaplotype (to produce
the pattern found in the exceptional 3 HbO-Arab
carrying chromosomes) has probably taken place much
later. This can also be supported by the 6:1 ratio between
the two different HbO-related haplotypic patterns. Additionally,
the fact that all HbO-Arab carrying chromosomes
displayed exclusively the 3´ subhaplotype pattern
(0)1(1001)1, implies that the above recombination
events were given the chance to occur for a limited time
only. If not so, one would expect to see more 3´ subhaplotypes
as the recombination rate within 3´ subhaplotype is
considerably high32. The latter is not true for the Greek
haplotype VI-related normal Pomak chromosomes, which
present three different 3´ subhaplotypes (types Greek VÉa,
VÉb and VÉc). These prospects are in keeping with the
results derived from the coalescent (HbO-Arab mutation
has emerged 2.883±2.471 years ago whereas the HbOArab
carrying Greek haplotype VIc recombined with
Universal haplotype Ia 366±561 years ago)50.
The worldwide-observed patterns of HBB frameworks
are considered to be ancient. The extremely low mutation
rate of the translatable region of the beta globin explains
the tendency that frameworks show to be conserved
throughout ages. Data concerning HBB frameworks are
not available in Greek population, but several international
studies can be taken into account37. Within this context,
finding the same framework CCTCT in close association
with the Greek VIa haplotype in all our HbO-Arab
cases as well as the all others already reported in the literature2,51
indicates a single origin of the mutation.
Our data can be compared with those of the last two
studies on the HbO-Arab related haplotypes over the
Mediterranean area:
The first2 has been carried out in two families, one of
Italian and one of Albanian origin. The HbO-Arab carriers
of the Italian family presented the Greek haplotype
VI 5´ subhaplotype pattern, while the HbO-Arab carriers
of the Albanian family the Benin one [0(?)0(0)00(?)1].
As both chromosomes presented the CCTCT framework
pattern, they thus can be explained as products of minor
recombination events evolved locally. The presence of the
African-specific 7.0 kb HpaI/HBB polymorphism, present
in both families, was not confirmed in our study as the
respective HpaI/3HBB(ii) polymorphism has not been
detected positive in any Pomak HbO-Arab carrier (Figure
2). Nevertheless, this is not necessarily controversial,
as it could be explained by a local recombination event.
The second51 was carried out in 20 carriers of the HbOArab
mutation from 9 families of non-Greek origin
(F.Y.R.O.M., Bulgarian, Turkish and Rom origin). All of
them were found to be associated with the Greek haplotype
VI 5´ subhaplotype and the CCTCT framework. The
HpaI/3´HBB(ii) positivity can be explained again by a local
recombination event within the Pomak HbO-Arab 3´
subhaplotype (0)1(1001)1 and a (?)1(?01?)1 one32,33.
Besides, the origin of HbO-Arab cannot be traced on the
populations from which the sample derived, as the Greek
haplotype VI as well as the CCTCT framework has never
been reported in their normal genes.
What Favoured the Increase of HbO-Arab Mutation
Frequency in Pomaks?
The presence of two ambiguous haplotypes in the
small, yet of unidentified origin, population of Pomaks is
an intriguing observation. As all other populations that
present these haplotypes have no geographical or historical
relation with Pomaks, a good explanation would be a
de novo formation and expansion in an environment of
intense genetic drift. Indeed, the mountainous area Pomaks
have inhabited from at least Byzantine Era in combination
with all available historical data, which confront
to their having very limited contacts with their neighbouring
populations (Greeks, Turks and Bulgarians), support
this hypothesis52. Besides, endogamy is socially acceptable
in Pomaks14.
The above prospects are in keeping with the extensive
linkage disequilibrium that is observed in Pomaks. Moreover,
the low number of observed haplotypes in relation
to expected alleles and the limited value of theta estimation
from k (èk) confronts to that Pomaks form a genetically
isolated population. The high gene diversity (H),
average gene diversity over loci (h), theta estimation from
H (èH) and mean intra- and inter-population number of
pairwise differences (ð) are attributed to the fact that two
of the most frequent haplotypes (Greek VI and Pomak I)
differ in many polymorphisms. As time to expansion (ô)
estimate leads to a high value (5.844), Pomaks are considered
to evolve slowly, as happens in a genetically isolated
population.
Under these circumstances, it is a matter of chance,
which of novel genetic events will be eventually selected
to dominate. The role of chance is enhanced when bottleneck
phenomena are superimposed. If, at last, the heterozygous
state of HbO-Arab carries a beneficial effect
for survival, its fixation will become easier and faster. It is
worth noticing that HbS that is absent from Pomaks, probably
because there had been no malaria selective pressure
in the region53. Nevertheless, haemoglobinopathies
have a balanced presence in areas without any obvious
reason17.
How and when the Novel 5 Subhaplotypes Found in
Pomaks were Formed?
Greek haplotype VI 5´ subhaplotype pattern may represent
the product of a recombination event between haplotypes
É and ÉÉ at the 9.4 kb sequence between HindIII/
HBG1 and HincII/HBBP1 polymorphisms. With a recombination
rate of 5x10-10 per nucleotide per generation, this
event can occur every 210000 meiotic events approximately33,54.
Considering that the Pomak population consists of
30,000 individuals, and that haplotypes I and II are detected
in 48.4% (15/31) and 6.5% (2/31) among normal
Pomak individuals respectively, the mean time interval
between two consequent recombination events in that
sequence is calculated to be close to 225 generations.
The possibility of a point mutation on the HincII/
3´HBBP1 site leading to the same result is less probable
as it is expected to happen at a rate similar to 7.4x10-9 per
nucleotide per generation (as calculated for HBB)48,55 or
every about 405400000 meiotic events approximately. In
a population of 30000 of which 48.4% present haplotype
I this event may happen every about 27900 generations.
The Pomak haplotype I 5´ subhaplotype pattern may
have resulted from a recombination event between the
common haplotypes ÉÉ or VI and III or ÉX at the 6.1 kb
long sequence between HindIII/HBG1 and HincII/
HBBP1 polymorphisms. This event, considered to occur
once every about 5000 generations, is expected to be far
more scarce that the recombinations mentioned above,
because haplotypes II, III, VI and IX are less common in
the Pomaks.
The hypothesis of a de novo nucleotide change in the
HincII/HBBP1 or HindIII/HBG1 restriction site on haplotypes
II and III, respectively, is again very improbable.
Given that haplotypes II and III are detected in 9.7% (3/
31) normal genes of Pomak origin, a population of 30000
meets an event like that every about 139000 generations.
The above mentioned suggestions are supported by
the fact that the only possible recombination site recognizable
by DNAsp 4.0 is between HindIII/HBG1 and
HincII/HBBP1 polymorphisms. Besides, they are in keeping
with the most probable estimated HbO-Arab age,
which is close to 144 generations (see Results).
Where do Pomaks come from?
The problem of tracing the origin of HbO-Arab mutation
could be efficiently transformed in tracing the origin
of their main carriers, Pomaks. The problem of the
origin of Pomaks has not been solved in a definite way
until our days. Despite that the dominant belief is that
Pomaks represent descendants of ancient Thracian
tribes52, there are still some scientists who believe that they
have originated from ancient African tribes or, less possibly,
from Mongolian tribes that inhabited Asia Minor and
Balkans after 7th century A.C.
The absence of the TaqI/3´HBG2 polymorphism in all
individuals of Pomak origin of the present study might
suggest an African origin30. This hypothesis could be further
supported by the selective presence of the abundant
in Pomaks Greek VI 5 subhaplotype pattern in small
numbers of African individuals (Table 4)17. On the other
hand, Pomaks lack common characteristics of the modern
African populations like the dominant 5´ subhaplotype
0(?)0(?)00(?)1, the HpaI/3HBB(ii) polymorphism
and the HbS mutation. Moreover, the main anthropological
characteristics of the Pomaks (white skin, blue eyes)
are far to be characterized as African.
Mongoloid characteristics are also absent from Pomaks.
Besides, the commonest HBB framework in Mongolia
(TGTCC) is totally absent from Pomaks37.
It is interesting to glance at the rooted tree constructed
by the use of pairwise differences based on data shown
in Table 4. The position of Pomaks in that tree is believed
to be somewhat misleading, as the presence of Greek haplotype
VI and Pomak haplotype I brings them closer to
Africans and Oceanians, respectively. To surpass this controversy,
a correction could be made under which the relative
frequencies of Greek haplotype VI and Pomak haplotype
I are equally divided and added to Universal haplotypes
I/II and II/III, respectively. This manipulation is
in keeping with the belief that the distinct Pomak haplotypes
were formed de novo, within the Pomak population,
as a result of intense genetic drift, by recombination
between the Universal haplotypes I, II and III. As a result,
a tree that brings Pomaks closer to Europeans and
Greeks (Figure
is constructed, enhancing the hypothesis
that Pomaks are descendants of ancient Thracian tribes.
Additionally, our own unpublished results confirmed that
Greeks are the only Europeans that share the absence of
TaqI/3´HBG2 polymorphism with Pomaks. Under this
point of view, Pomaks might carry genes that took part in
ancient recombination events along with African genes.
Reconstruction of the Puzzle of HbO-Arab
Mutation History
Be this as it may, we hypothesize that the formation of
the Pomak É 5´ subhaplotype occurred in the ancestors of
this population several thousand years ago in a similar
way while the ancestors of the present day Pomaks had
already been separated as a distinct population. Within
this group and for a relatively short period only, the novel
5´ subhaplotypes recombined with the standard 3´ subhaplotypes
(of Universal haplotype I, II, or III) to form the
final haplotypes found in the normal Pomak chromosomes.
We further hypothesize that the subsequent spread of
HbO-Arab carrying chromosomes over the Mediterranean
basin and the Middle East took place was effected either
during the Alexander the Great expansion or the Ottoman
Empire; minor local recombination events may have
Figure 8. Tree suggesting possible evolutionary relationship between
populations of Table 4 (after equal distribution of Pomakspecific
haplotypes Greek VI and Pomak I to their hypothetical
ancestor haplotypes I/II and II/III, respectively).
occurred later, without loosing the original characteristics
of the mutation.
Summing up, Hb O-Arab was first arisen in Pomaks,
on the rare Greek VIc haplotype and CCTCT framework
about 2000 years ago and dispersed throughout Mediterranean
and Middle East countries, where minor recombination
events took place.
ACKNOWLEDGEMENTS
The present study was financially supported by the
Greek National Scholarship Foundation (I.K.Y.). All authors
express their thanks to Ms Kally Simitopoulou and
Ms Andrea Zatkova from University of Thrace, Department
of Anthropology and Ms Irene Stratidaki and Mr
Nick Kountourakis from University of Crete, Institute of
Technology and Research for their excellent technical
support.
