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Comparison of lactase persistence polymorphism in ancient and present-day Hungarian populations.

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AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 145:262–269 (2011)
Comparison of Lactase Persistence Polymorphism in
Ancient and Present-Day Hungarian Populations
Dóra Nagy,1,2* Gyöngyvér Tömöry,1 Bernadett Csányi,1 Erika Bogácsi-Szabó,1 Ágnes Czibula,1
Katalin Priskin,1 Olga Bede,2 László Bartosiewicz,3 C. Stephen Downes,4 and István Raskó1
1
Institute of Genetics, Biological Research Centre of Hungarian Academy of Sciences, Szeged H-6726, Hungary
Department of Pediatrics, University of Szeged, Szeged H-6725, Hungary
3
Archeological Institute of the Hungarian Academy of Sciences, Budapest H-1014, Hungary
4
School of Biomedical Sciences, University of Ulster, Coleraine BT521SA, Northern Ireland
2
KEY WORDS
lactose intolerance; ancient DNA; 10th–11th century bones; mitochondrial DNA
ABSTRACT
The prevalence of adult-type hypolactasia varies ethnically and geographically among populations. A C/T–13910 single nucleotide polymorphism
(SNP) upstream of the lactase gene is known to be associated with lactase non-persistence in Europeans. The
aim of this study was to determine the prevalence of lactase persistent and non-persistent genotypes in current
Hungarian-speaking populations and in ancient bone
samples of classical conquerors and commoners from the
10th–11th centuries from the Carpathian basin; 181
present-day Hungarian, 65 present-day Sekler, and 23
ancient samples were successfully genotyped for the C/T13910 SNP by the dCAPS PCR-RFLP method. Additional
mitochondrial DNA testing was also carried out. In ancient Hungarians, the T-13910 allele was present only in
11% of the population, and exclusively in commoners of
European mitochondrial haplogroups who may have
been of pre-Hungarian indigenous ancestry. This is despite animal domestication and dairy products having
been introduced into the Carpathian basin early in the
Neolithic Age. This anomaly may be explained by the
Hungarian use of fermented milk products, their greater
consumption of ruminant meat than milk, cultural differences, or by their having other lactase-regulating
genetic polymorphisms than C/T-13910. The low prevalence of lactase persistence provides additional information on the Asian origin of Hungarians. Present-day
Hungarians have been assimilated with the surrounding
European populations, since they do not differ significantly from the neighboring populations in their possession of mtDNA and C/T-13910 variants. Am J Phys
Anthropol 145:262–269, 2011. V 2011 Wiley-Liss, Inc.
Lactose intolerance (adult-type hypolactasia, lactase
non-persistence) is a common trait worldwide, which
varies between populations, both ethnically and geographically. The activity of the lactase enzyme, which
facilitates the digestion of milk-derived lactose,
decreases after weaning in most humans, but persists in
some; when it does not persist, lactose intolerance
results. The prevalence of adult-type lactose intolerance
among Caucasian populations in Europe varies between
3 and 70%, in contrast with up to 100% among Asians
(Sahi, 1994; Swallow, 2003). European lactase persistence is associated with a single nucleotide polymorphism
(SNP), 13910 base pairs (bp) upstream of the lactase
gene, in one of the introns of the MCM6 gene (Enattah
et al., 2002), which appears to function also as a regulatory region for lactase transcription (Olds and Sibley,
2003; Troelsen et al., 2003). The CC213910 genotype is
associated with lactase non-persistent, CT213910 and
TT213910 genotypes with lactase persistent phenotypes
(Enattah et al., 2002).
Several hypotheses have been proposed to explain the
geographic distribution of lactase persistence. The most
commonly acknowledged view is the ‘‘gene-culture coevolution/culture-historical’’ hypothesis, which supposes
that lactase persistence most likely originated as a
mutation in populations which used milk or milk products from domesticated animals as an important source
of adult nutrition (Simoons, 1970; Beja-Pereira et al.,
2003). A strong positive selection is then supposed to
have occurred for the lactase persistent phenotype, from
the ancestral lactase non-persistent phenotype, around
5,000–10,000 years ago (Bersaglieri et al., 2004). An alternative, the ‘‘reverse cause’’ hypothesis, is that some
populations were pre-adapted for the use of milk as an
adult food, having acquired a substantial frequency of
the T213910 allele by random drift, before they took up
dairy farming (Burger et al., 2007).
The population we are concerned with here, Hungarian pastoralist nomads, after a period of migration from
much further east, entered Europe as seven major tribes
that invaded the Carpathian basin from across the encircling mountains around 895 AD. The Hungarian language belongs to the Finno-Ugric branch of the Uralic
family, a diverse group, living in Northern Europe and
in the regions west and east of the Ural. During the
C 2011
V
WILEY-LISS, INC.
C
Additional Supporting Information may be found in the online
version of this article.
Grant sponsor: Hungarian National Research and Development
Programs; Grant numbers: OM-00050/2004.
*Correspondence to: Dóra Nagy, Institute of Genetics, Biological
Research Centre of Hungarian Academy of Sciences, POB 521,
Szeged H-6701, Hungary. E-mail: nagydor@gmail.com
Received 31 August 2010; accepted 13 December 2010
DOI 10.1002/ajpa.21490
Published online 1 March 2011 in Wiley Online Library
(wileyonlinelibrary.com).
LACTASE PERSISTENCE IN ANCIENT AND RECENT HUNGARIANS
migration they might have been in contact with FinnoUgric populations, Kazars, Petchenegs, Bolgars, Savirs,
Kabars, and Alans living in the northwestern and southwestern regions of the Ural and in the Caucasus. The
Carpathian basin had been settled for thousands of
years before the arrival of the Hungarians, by Dacians,
Romans, Sarmatians, Goths, Huns, Avars, Slavs, and
many others: it is probable that on the eve of the Hungarian conquest the majority of the indigenous population were Slavic (Tömöry et al., 2007).
The aim of this study was to evaluate the prevalence
of C/T213910 genotypes in remains from the Hungarian
population of the 10th–11th centuries, as compared with
current Hungarian-speaking populations. Random samples were evaluated, and our results are analyzed relative to other populations, which are believed to have
been in contact with Hungarians during the migratory
period, and after the settlement of the Hungarians in
the Carpathian basin.
MATERIALS AND METHODS
Present-day samples
In this study, unrelated adults from different parts
of Hungary and Transylvania were involved, 181 Hungarians and 65 Seklers (or Székely, an outlying minority
Hungarian-speaking population in eastern Transylvania,
supposedly descendants of one of the seven invading
tribes). The individuals gave their informed consent to the
study, which was approved by the local ethical committee.
DNA extraction from present-day samples. Genomic
DNA was extracted from the root section of individual
hairs or buccal smears, using the Chelex-based method
(Walsh et al., 1991).
Mitochondrial DNA (mtDNA) testing of present-day
samples. The hypervariable region I (HVSI) of the control region and, if necessary, HVSII and coding regions
of the mtDNA were analyzed either by sequencing or by
the PCR-RFLP method to elicit the mtDNA haplogroup
of each sample. The method was identical to that
described by Tömöry et al. (2007). Polymorph positions of
the mtDNA were identified using the revised Cambridge
Reference Sequence (Andrews et al., 1999). The samples
were mtDNA haplogrouped based on mutational patterns summarized by Tömöry et al. (2007). All presentday Sekler samples were mitochondrially haplogrouped.
Seventy-one of the 181 present-day Hungarian samples
were randomly selected for mtDNA testing.
C/T213910 genotyping of present-day samples. The
derived cleaved amplified polymorphic sequence method
(dCAPS) was used to analyze the genotypes. A restriction
enzyme recognition site including the SNP was introduced
into the PCR product by the forward primer containing a
mismatch to template DNA, using the dCAPS Finder 2.0
program (http://helix.wustl.edu/dcaps/dcaps.html). The
forward primer was: 50 -GGCAATACAGATAAGATAATGG
AG-30 ; reverse primer: 50 -CCTATCCTCGTGGAATGCA
GG-30 ; mismatching nucleotide underlined (Nagy et al.,
2009). PCR amplification was carried out in 40 ll reactions containing 13 Ampli Taq Gold Buffer, 6 lM of each
primer, 200 lM of each deoxynucleotide-triphosphate
(dNTP), 2.5 mM MgCl2, 20 ng DNA extract, and 1 U
AmpliTaq Gold Polymerase (Applied Biosystems, CA). The
amplification protocol: 6 min at 948C, 35 cycles of 938C
263
for 45 s, 548C for 45 s, 728C for 60 s, and final extension at
728C for 5 min.
Seven microliter of the 119 bp-modified PCR product
was subjected to NlaIV and HinfI (Fermentas, Ontario,
Canada) restriction enzyme digestions, with 5 U restriction enzyme and 13 reaction buffer in a reaction volume
of 20 ll, to detect the C213910 and T213910 alleles. NlaIV
enzyme cleavage resulted in two fragments (96 bp and
23 bp) for the C allele, while HinfI digestion yielded a
97 bp and a 22 bp fragment for the T allele. The
enzyme-cleaved PCR products were run on 8% native
polyacrylamide gel and visualized after ethidium bromide staining by UV transillumination with the UVP
BioImaging System (Upland, CA).
Ancient samples
Forty-two bone samples in an excellent state of biomolecular preservation, originating from burials in the
period of the Hungarian conquest were included in the
analysis. The samples, provided by the Archeological
Institute of the Hungarian Academy of Sciences, had been
excavated in cemeteries from the 10th–11th centuries
from different regions of the Carpathian basin (Table
S1a). Both the burial sites and the bones were archaeologically and anthropomorphologically well-defined. The
groups we have called classical conquerors and commoners were distinguished on the basis of the grave findings. Classical Hungarian conquerors were those excavated from rich graves, containing a horse skull, harness,
arrow- or spear-heads, mounted belts, braided ornaments
and earrings. Commoners were found in graves with
impoverished burial remains. All 42 ancient bone samples
had previously yielded reproducible mtDNA [bone samples were identical to those analyzed by Tömöry et al.
(2007) and to not yet published samples; Table S1a]. Processing of ancient samples was carried out according to the
study of Kalmár et al. (2000), Tömöry et al. (2007), and
Csányi et al. (2008).
Bone powdering and DNA extraction from ancient
samples. The surface of the bones (femurs) was washed
with diluted bleach and distilled water and was treated
with UV-C irradiation at 1 J/cm2 for 30 min. A 2 cm 3 3
cm portion was cut from each bone epiphysis and the
surface of these portions was washed with bleach and
removed (at least 2–3 mm deep) with a UV-C treated
sterilized sand disk. The bone portion was then treated
at each side with UV-C light at 1 J/cm2 for 30 min and
ground into fine powder by using mineralogy mill
(Retsch MM301; Haan, Germany) and then stored in
sterile tubes at 48C. Bone powder (1.3–1.5 g) was suspended in 10 ml EDTA and incubated overnight at 378C
with continuous vertical rotation. The samples were centrifuged (2000g for 15 min), EDTA was removed and the
sediment was resuspended in 10 ml EDTA everyday for
3–5 days. The sediment was suspended in 1.8 ml of
extraction buffer, incubated overnight at 378C with continuous vertical rotation, and centrifuged at 12,000 rpm
for 10 min. The supernatant, containing the DNA, is
stored at 2208C.
DNA isolation from ancient samples. Standard isolation methods were used as described by Kalmár et al.
(2000) and alternatively when needed, a modified
method incorporating the DNeasy Tissue Kit (Qiagen,
Valencia, CA) was used. In this modified method, DNA
was isolated from 350-ll bone extract, by treatment with
American Journal of Physical Anthropology
264
D. NAGY ET AL.
350 ll 4 M NH4-acetate and 700 ll 96% EtOH at 2708C
for 10 min. The mixture was transferred into DNeasy
Mini spin column and centrifuged at 6000g for 1 min.
The column was washed twice and DNA was eluted in a
final volume of 40 ll (Tömöry et al., 2007).
Mitochondrial DNA (mtDNA) testing of ancient
samples. All ancient samples were previously tested for
mtDNA haplogroups (Tömöry et al., 2007; Table S1b).
C/T213910 genotyping of ancient samples. After the
successful and contamination-free mtDNA amplification,
C/T213910 genotyping was carried out on the ancient samples. The primers and restriction enzymes were identical
to those used in the reactions of the present-day samples.
The standard amplification reaction of the ancient
samples contained 10 ll of bone isolation, 200 lM of
each of the dNTPs, 25 pmol of each primer, 2.5 mM
MgCl2, 13 Colorless GoTaq Flexi Buffer, and 1.25 U
GoTaq Hot Start Polymerase (Promega, Wisconsin) in a
total reaction mixture volume of 50 ll. The amplification
protocol was: 948C for 6 min, 10 cycles of 30 s at 938C, 40 s
at 568C, 40 s at 728C, 40 cycles of 30 s at 938C, 40 s at
548C, 40 s at 728C, and a final extension at 728C for 5 min.
Twenty of the 42 ancient samples yielded DNA in the
PCR reaction. The remaining 22 samples were subjected
to a modified improved primer extension preamplification (mIPEP) method (Hanson et al., 2005; Csányi et al.,
2008) to enhance the efficiency of the amplification. Ten
microliter aliquots of mIPEP products were used in
subsequent C/T213910 genotyping. Three further ancient
samples were successfully typed (Table S1b).
Ten microliter of the PCR product was subjected to
restriction enzyme digestions as described in connection
with the present-day samples (Fig. S1).
the results. Those samples were accepted that had consistent results in all successful PCR reactions and had different results from the haplogroup and genotype results
of the researchers who analyzed the samples. Extraction,
preamplification, amplification, and digestion blanks
(with no bone powder, template DNA) were used as negative controls in each reaction to screen for contaminants
entering the process at any stage. Positive controls (CC,
CT, and TT213910) were also used in each digestion.
Due to DNA degradation, primers were designed to
amplify short sequences of templates during mtDNA
testing (Tömöry et al., 2007) and C/T213910 genotyping
(119bp-long PCR product).
To prove the authenticity of ancient human DNA further, DNA was isolated from an ancient horse remain,
excavated from one of the human burial sites, and
amplified with both the horse-specific (forward: 50 -CAC
CATACCCACCTGACATGCA-30 and reverse: 50 -GCTGA
TTTCCCGCGGCTTGGTG-30 ) and the human-specific
C/T213910 primers. Only the horse-specific primers
resulted in amplification product.
Statistical analysis
The GraphPad Prism version 4.00 for Windows software (GraphPad Software, San Diego, CA) was used.
The Fisher exact test was performed to compare the C/
T213910 genotypes in the ancient and present-day populations. Deviation from Hardy-Weinberg equilibrium was
calculated (Rodriguez et al., 2009) in present-day and
ancient Hungarian populations concerning the C/T213910
genotypes. A probability level P \ 0.05 was considered
to be statistically significant.
RESULTS
Contamination prevention and authentication
To prevent any possible contamination with modern
DNA, strict precautions were taken during each step of
the ancient sample preparation, as described by Tömöry
et al. (2007). The 11 persons, who participated in the
sample processing or worked in the labs, were mtDNA
tested and C/T213910 genotyped (Table S2). The number
of persons involved in the processing was minimized as
much as possible in order to prevent contamination. All
steps of sample processing (bone powdering, DNA extraction, preamplification, amplification and post-PCR analysis) were carried out wearing appropriate protective
clothing (gloves, face mask, hair net, glasses, and laboratory coats) in separate rooms dedicated for ancient DNA
work and free of other molecular work. All workspaces
and appliances were cleaned with bleach and subsequently irradiated with 1 J/cm2 UV-C light for 2 h before
use. All solutions used were filtered and subsequently
irradiated with UV-C light for 30 min. During all steps
Universal Fit Filter Tips (Corning Incorporated, Lowell,
MA) were used for pipetting. PCR and Eppendorf tubes
were sterilized before use by autoclaving.
The surface of the bone samples was cleaned and
removed as described above in order to prevent possible
contamination. Bones were powdered independently by at
least two researchers with different mtDNA haplogroups
and C/T213910 genotypes, at least two times each. In each
case, two independent DNA extractions were carried out,
and at least two successful mIPEP and/or PCR amplifications were performed from each extract and/or mIPEP
product to assess the reproducibility and authenticity of
American Journal of Physical Anthropology
The genotyping of the C/T213910 autosomal SNP was
successful in 23 ancient bone samples (13 classical conquerors, nine commoners, and one not determined). The
C/T213910 genotype, and the mtDNA haplogroup results
and the features of the bone samples are shown in Table
S1a and S1b. The prevalence of the C/C213910, C/T213910,
and T/T213910 genotypes among the 23 ancient Hungarians was 87%, 4%, and 9% (Table 1); as compared with
39%, 50%, and 11% among 181 present-day Hungarians;
and 29%, 62%, and 9% among 65 present-day Seklers
(Table 2). The allele frequencies associated with lactase
persistence (T213910) in the groups of ancient, presentday Hungarians and present-day Seklers were 10.9%,
35.9%, and 40%, respectively. Although all 13 classical
conquerors had C/C213910 genotype, three of the commoners displayed C/T213910 (11%) and T/T213910 genotypes (22%) (Table 1). The T213910 allele frequency was
28% among the commoners.
The additional mtDNA testing identified six major
mtDNA haplogroups (H, U, T, N1a, JT, X) among Hungarian conquerors, six among commoners from the time
of the conquest (H, HV, M, R, T, U) and 13 (H, HV, I, J,
K, JT, M, R, T, U, V, W, X) among present-day Hungarian-speaking populations. The three ancient samples
with a lactase persistent genotype were all commoners
and all displayed haplogroup H, which is the most common in Europe (Richards et al., 1998). Two of these samples showed TT213910 genotype. Although they both displayed haplogroup H, their mutations in the HVSII and
coding regions of the mtDNA were not identical which
excludes their maternal relationship. They were buried
265
LACTASE PERSISTENCE IN ANCIENT AND RECENT HUNGARIANS
TABLE 1. Distribution of mtDNA haplogroups and C/T213910 genotypes among ancient Hungarians
Number of classical
conquerors (%)
Number of
commoners (%)
CC213910 CT213910 TT213910
mtDNA
13 (100)
haplogroup
H
4
U
3
T
2
N1a
2
JT
1
X
1
HV
0
R
0
M
0
J
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Total
CC213910 CT213910 TT213910
13 (100)
4
3
2
2
1
1
Total number of
ancient Hungarians (%)
(31)
(23)a
(15)c
(15)
(8)
(8)
0
0
0
0
6 (67)
1 (11)
2 (22)
0
2
1
0
0
0
1
1
1
0
1
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
Total
9 (100)
3 (33.5)
2 (22.5)b
1 (11)d
0
0
0
1 (11)
1 (11)
1 (11)
0
CC213910 CT213910 TT213910
20 (87)
1 (4)
2 (9)
4
5
3
2
1
1
1
1
1
1e
1
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
Total
23 (100)
7
5
3
2
1
1
1
1
1
1
(31)
(23)
(13)
(9)
(4)
(4)
(4)
(4)
(4)
(4)
mtDNA, mitochondrial DNA. Further types were identified within haplogroup U:
a
U4 in two cases,
b
U3 and U5a1 in one case each; haplogroup T:
c
T3 in 1 case,
d
T2 in 1 case; haplogroup J:
e
J2 in 1 case, the social status of this sample was not classified.
TABLE 2. Distribution of mtDNA haplogroups and C/T213910 genotypes among present-day Hungarian-speaking populations
Number of present-day Hungarians (%)
CC213910
mtDNA haplogroup
H
U
T
J
K
V
HV
W
R
X
M
JT
I
71 (39)
30 (42)
13
0
3
5
4
2
2
0
1
0
0
0
0
CT213910
90 (50)
37 (52)
15
6
2
3
3
1
0
3
2
2
0
0
0
TT213910
20 (11)
4 (6)
1
0
0
0
0
1
1
0
0
0
1
0
0
Number of present-day Seklers (%)
Total
181
71
29
6
5
8
7
4
3
3
3
2
1
a
(100)
(100)b
(41)
(8)c
(7)e
(11)g
(10)
(6)
(4.2)
(4.2)
(4.2)
(3)
(1.4)
0
0
CC213910
CT213910
TT213910
19 (29)
19 (29)
6
2
4
1
3
0
2
0
0
1
0
0
0
40 (62)
40 (62)
16
8
6
3
4
0
0
1
0
0
0
1
1
6 (9)
6 (9)
2
1
1
1
0
0
0
1
0
0
0
0
0
Total
65
65
24
11
11
5
7
2
2
1
1
1
(100)a
(100)b
(36)
(16.5)d
(16.5)f
(8)h
(11)
0
(3)
(3)
0
(2)
0
(2)
(2)
mtDNA, mitochondrial DNA.
a
Number of all C/T213910 genotyped samples.
b
Number of all mtDNA tested and C/T213910 genotyped samples. Further types were identified within haplogroup U:
c
U5, U5a, and U5b in one case each and U4 in two cases,
d
U3, U4, and U5a1a in one case each and U5a1 in five cases; haplogroup T:
e
T1 and T3 in one case each and T2 in two cases,
f
T2, T2b, T3, and T5 in one case each and T1a in five cases; haplogroup J:
g
J1 and J1a in one case each and
h
J2 in one case.
in graves of different location and time which may further support that these individuals were not related to
each other (Table S1a and S1b). Haplogroup N1a (indicative of a Near Eastern, Asian origin—Haak et al., 2005)
and haplogroup M (indicative of an Asian origin—MacaMeyer et al., 2001) were present in 9% and 4% of the
ancient Hungarians, but absent or very rare in the present-day Hungarian and Sekler populations. The high
prevalence of haplogroup U (23% of the ancient samples), and especially the haplogroup U4, is characteristic
of Finno-Ugric populations and populations in southeastern Europe and western Siberia (Richards et al.,
1998; Bermisheva et al., 2002) (Table S3).
Significant difference was found in the C/T213910
genotypes and allele frequencies between the ancient
Hungarian conquerors and the present-day Hungarianspeaking populations. No significant difference was
found between the present-day Hungarian-speaking populations and ancient Hungarian commoners (Table 3).
Table S4 presents the frequencies of the T213910 allele,
digesters and non-digesters in present-day populations of
the Uralic linguistic family and populations which according to historical accounts (Vékony, 2002) might well have
been in contact with ancient Hungarians during their
westward migratory period, and after the settlement of
the Hungarians in the Carpathian basin. Present-day
Hungarian-speaking populations exhibit a similar prevalence of the T213910 allele to those in neighboring countries, such as Austria, the Czech Republic, Slovenia, and
Germany (Högenauer et al., 2005; Gerbault et al., 2009;
American Journal of Physical Anthropology
266
D. NAGY ET AL.
TABLE 3. Comparison of the distribution of C/T213910
genotypes and allele frequencies in present-day and ancient
Hungarian-speaking populations
Present-day
Hungarians
All ancient Hungarians
Genotype
P \ 0.0001
Allele frequency
P 5 0.0004
Classical conquerors
Genotype
P \ 0.0001
Allele frequency
P \ 0.0001
Commoners
Genotype
P 5 0.1617
Allele frequency
P 5 0.6169
Present-day
Seklers
Classical
conquerors
P \ 0.0001
P 5 0.0002
–
–
P \ 0.0001
P \ 0.0001
–
–
P 5 0.0537
P 5 0.4399
P 5 0.0545
P 5 0.0079
P \ 0.05 is considered to be statistically significant.
GLAD). In contrast, the group of all ancient Hungarians
displayed a significantly lower prevalence. Although the
prevalence of the T213910 allele in the subgroup of ancient
commoners was similar to that of the present-day
Hungarians, North-west Russians, Austrians, Slovaians,
Chechs, and Germans; in the subgroup of classical conquerors it corresponded well with the prevalence of ObUgric present-day populations, such as Khantys, or Maris,
and certain Central-Asian and Turkish populations
(Lember et al., 1995; Kozlov et al., 1998; Enattah et al.,
2007; Sun et al., 2007; GLAD) (Table S4).
Significant deviation from Hardy Weinberg Equilibrium was calculated in the group of ancient Hungarians
(P \ 0.001) and in the subgroups of commoners (P \
0.05). No significant deviation was found in the presentday Hungarian population (P [ 0.10).
DISCUSSION
We succeeded in genotyping the C/T213910 autosomal
SNP in an unprecedented number of ancient bone samples. It has been proposed (Burger et al., 2007) that
allele determination by PCR of ancient samples may be
misleading on account of the possibility of allele dropout:
the ancient DNA may be so sparse that random distribution of the alleles in the PCR reaction mixture may
result in only one allele being present. If this happens, a
heterozygote would be falsely reported as a homozygote.
However, this effect would only strengthen our conclusions. Dropout might remove both alleles from the reaction mixture, so nothing would appear in our dCAPSPCR results. The much more probable loss of one allele
from homozygotes would not affect the results. Random
loss of one allele or the other from heterozygotes would
reduce the proportion of apparent heterozygotes, and
increase apparent homozygotes, both CC and TT. But
our analysis detected no TT homozygotes in classical
conquerors, and unusually few in commoners. If allele
dropout has occurred, that would not affect the first
result, and would imply that the true level of TT homozygotes in commoners is even lower than our estimate.
Beside this explanation we made efforts to reduce the
possible allele dropout. High-quality and -quantity bone
powder was used to optimize DNA extraction; preamplification method (mIPEP) was applied to increase the
quantity of template DNA before PCR reaction; the number of amplification cycles were increased to reach the
detection limit of the machine without strong artifacts;
and amplification results were concluded from several,
consistent PCR reactions as it is suggested in studies on
American Journal of Physical Anthropology
ancient DNA by Burger et al. (1999) and Hummel
(2003).
To interpret our results correctly, we favor a multidisciplinary approach, including data on genetic testing of
mtDNA and Y chromosomes, Hungarian history, and direct
and indirect evidence from archaeology and ethnography.
The ability to digest lactose into adulthood is one of
the traits that developed in mankind under strong selection pressure (Bersaglieri et al., 2004). The selection
forces could have been the nutritional benefit, the
water and electrolyte contents of the milk or the
improved calcium absorption after milk consumption.
The ‘‘gene-culture co-evolution’’ hypothesis (Simoons,
1970; McCracken, 1971; Holden et al., 2002) postulates
that the selection force was the nutritional benefit in
those nomad pastoral populations which originally used
processed, lactose-low milk products (cheese, yoghurt,
kumis, etc) for which the lactase persistent allele was
not advantageous, but who subsequently had strong
selective pressure for those who could also drink raw
milk. Such selection appears to have operated substantially later than the earliest domestication of ruminants
in the Middle East, around 8000 BC, and later than the
first use of milk, as assessed via the d13C values of fatty
acids in pottery vessels; that is, around 7000 BC in the
Near East and south-eastern Europe (Evershed et al.,
2008). Though only small-scale dairying existed between
7000 and 4000 BC, it increased in the period 4000–3000
BC (Craig et al., 2005). Beja-Pereira et al. (2003) have
argued that the ability for adults to consume milk coevolved with the ability of dairy cattle to give high milk
yields; and that adult lactose tolerance evolved in northern Neolithic Europe, where the relatively cool climate
allows unfermented milk to be stored for a while.
There is good evidence that Early Neolithic farmers
lacked adult lactose tolerance. Burger et al. (2007) concluded that the lactase persistent T213910 allele was not
observed in human remains from European Neolithic
and Mesolithic sites (5840-2267 BC), whereas it was in
one Mediaeval sample (AD 400-600). Population studies
(Enattah et al., 2007) are consistent with the T213910 allele having arisen twice, once between 5 and 12 thousand years ago in the haplotype which is now dominant
in Europe, and once 1400–3000 years ago in a region
north of the Caucasus and west of the Urals. Itan et al.
(2009) used a demic computer simulation model to
explore the spread of European lactase persistence, and
concluded that it originated in central Europe, in a
region including modern Hungary, about 5500 BC. The
molecular evidence, then, is in favor of gene/culture coevolution, not of the reverse-cause hypothesis
How do our results fit into this picture? Present-day
Hungarian-speaking populations and ancient commoners
exhibit a similar prevalence of the T213910 allele to those
in neighboring countries, such as Austria, the Czech
Republic, the Ukraine, and Slovenia. In contrast, the ancient classical conquerors displayed a significantly lower
prevalence, which corresponds well with those of present-day populations of the Uralic linguistic family, such
as the Khantys, Mansis and Maris, and certain CentralAsian and Turkish populations (Table S4). This is consistent with the original Hungarian invaders, having
roots far to the east of modern Hungary, being a minority in the Carpathian basin, further diluted in the subsequent turbulent history of that area. This is supported
by previous Hungarian studies on mitochondrial haplogroups and Y chromosome (Tömöry et al., 2007; Csányi
LACTASE PERSISTENCE IN ANCIENT AND RECENT HUNGARIANS
et al., 2008), which showed the genetic assimilation of
the present-day Hungarians with their geographical
neighbors, but also a significant Asian influence on the
genetics of the Hungarian conquerors, from their Siberian origins and from the genetic effect of those populations with whom the ancient Hungarians came into
contact during their westwards migrations, such as the
Kazars, Petchenegs, Bolgars, Savirs, and Iranians.
Among the archaeological remains from the period of
the Hungarian conquest, the absence of the T213910 allele
in the classical conquerors, but not in the commoners,
might suggest a hierarchical difference in nutrition. It
might also be due to some of the commoners being
descendants of the pre-existing inhabitants of the Carpathian basin, who lived a settled life and had a different
dairy culture from that of the Hungarian conquerors.
There is some evidence for a change in stockraising
practices at the time of the Hungarian conquest. In
archaeological sites in the Carpathian basin from the
Sarmatian (1st–4th centuries AD), Slavic and Avar (5th–
9th centuries AD), and Hungarian conquest period (9th–
10th centuries AD) cattle remains dominated animal
bone assemblages; but during the Hungarian conquest, a
small increase occurred in the number of horse bones
and a small decrease in the number of cattle bones. The
fragmentation of the bones indicated a butchering process; the change could therefore be due to differential
consumption of horse and cattle meat (Batrosiewicz,
2003). Nevertheless, milk products must have been
renewable sources of food for the ancient Hungarian pastoralists; mobile pastoralists collect milk from their
herds, including mares (Batrosiewicz, 2003; Outram et
al., 2009).
The absence of adult lactose tolerance in the ancient
Hungarians we have studied is compatible with their
milking their herds, given milk fermentation (Myles et
al., 2005; Ingram et al., 2009). While fresh milk was the
basic dairy food in Scandinavia, a situation in accord
with the cold climate and good sanitation, in south-eastern Europe and in south-western Asia processed foods
prepared from soured milk were preferred. Milk was less
appetizing as a fresh warm beverage than as fermented
foods (Kosikowski, 1981; Outram et al., 2009). The
lactose content of fresh milk is 4.42–5.15 g/g% in
cattle (Cerbulis et al., 1974; Miglior et al., 2006), 4.66–
4.82 g/g%in the goat (Baldi et al., 2002; Contreras et al.,
2009), 4.57–5.40 g/g% in the sheep (Fuertes et al., 1998;
Addis et al., 2005), and 6.91–7.04 g/g% in the horse
(Caroprese et al., 2007). Since the lactose content can be
reduced by 50–60% by bacterial fermentation (Kilara et
al., 1975), processed milk products have no or low lactose
contents (ranging 0–3.7 g/g%). Lactose malabsorbers
reported fewer or no symptoms after consuming fermented milk products (Alm, 1982; Savaiano et al., 1987).
Such processing of milk is of considerable antiquity, to
judge by the archaeological evidence. The presence of
degraded milk fats (high abundances of C16:0 and C18:0
fatty acids) and lipid pyrolysis products (mid- and longchain ketones) in some archaeological ceramics suggests
that the dairy products were heated, perhaps as part of
their processing (Craig et al., 2005; Evershed et al.,
2008). This is supported by the fact that a high frequency of ruminant milk lipids has been detected from
such ceramics, whereas raw milk lipids (high abundances of C4:0 to C12:0 fatty acids) are rapidly destroyed by
burial (Evershed et al., 2008; Copley et al., 2003). Hungarian pastoralists, averse to drinking fresh milk, would
267
have had little or no selection for lactose tolerance in
earlier millennia.
Alternatively, the low prevalence of European-type
lactase persistence in ancient Hungarians may be due to
their having one or more non-European polymorphisms
in the lactase regulatory regions. Recent studies summarized by Ingram et al. (2007), Enattah et al. (2008),
and by Itan et al. (2010), have revealed other alleles that
can also produce adult lactose tolerance: G213907,
G213915, and C214010 in East African populations, a compound G213915/C23712 allele in Saudi and other Middle
Eastern populations. Some cases of undoubted lactose
tolerance have not yet been identified with any
known allele in the control regions sequenced (Itan et
al., 2010); notably, lactose tolerant northern Chinese
whose closeness to the central Asian steppe may allow
some common ancestry with early Hungarians lack the
T213910 allele, and must have some other unidentified
allele instead (Sun et al., 2007). It is therefore possible
that ancient Hungarians were lactose tolerant despite
their lack of the characteristic European allele. In view
of the fairly high proportion of European Y chromosome
and mitochondrial haplogroups among Hungarian conquerors (Tömöry et al., 2007; Csányi et al., 2008), that is
perhaps unlikely: and in view of our ignorance of the
non-European allele that might be involved, it is premature to search for it in ancient Hungarian samples.
In conclusion, in ancient Hungarians, the T213910 allele
was present only in 11% of the population, and exclusively
in commoners of European mitochondrial haplogroups
who may have been of pre-Hungarian indigenous ancestry. This is despite animal domestication and dairy products having been introduced into the Carpathian basin
early in the Neolithic Age. This anomaly may be explained
by the Hungarian use of fermented milk products, their
greater consumption of ruminant meat than milk, cultural differences, or by their having other lactase-regulating genetic polymorphisms than C/T213910. The low prevalence of lactase persistence provides additional information on the Asian origin of Hungarians. Present-day
Hungarians have been assimilated with the surrounding
European populations, since they do not differ significantly from the neighboring populations in their possession of mtDNA and C/T213910 variants.
ACKNOWLEDGMENTS
The authors thank Mrs. Mária Radó and Mrs. Gabriella Leho†cz for their skilled technical assistance, and
Balázs Mende and Péter Langó for the archaic samples.
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