High frequency of alkaptonuria in Slovakia: evidence for the appearance of multiple mutations in HGO involving different mutational hot spots.

Abstract

Alkaptonuria (AKU) is an autosomal recessive disorder caused by the deficiency of homogentisate 1,2 dioxygenase (HGO) activity. AKU shows a very low prevalence (1:100,000-250,000) in most ethnic groups. One notable exception is in Slovakia, where the incidence of AKU rises to 1:19,000. This high incidence is difficult to explain by a classical founder effect, because as many as 10 different AKU mutations have been identified in this relatively small country. We have determined the allelic associations of 11 HGO intragenic polymorphisms for 44 AKU chromosomes from 20 Slovak pedigrees. These data were compared to the HGO haplotype data available in our laboratory for >80 AKU chromosomes from different European and non-European countries. The results show that common European AKU chromosomes have had only a marginal contribution to the Slovak AKU gene pool. Six of the ten Slovak AKU mutations, including the prevalent G152fs, G161R, G270R, and P370fs mutations, most likely originated in Slovakia. Data available for 17 Slovak AKU pedigrees indicate that most of the AKU chromosomes have their origins in a single very small region in the Carpathian mountains, in the northwestern part of the country. Since all six Slovak AKU mutations are associated with HGO mutational hot spots, we suggest that an increased mutation rate at the HGO gene is responsible for the clustering of AKU mutations in such a small geographical region.

Full text

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Am. J. Hum. Genet. 67:1333–1339, 2000
1333
Report
High Frequency of Alkaptonuria in Slovakia: Evidence for the Appearance
of Multiple Mutations in HGO Involving Different Mutational Hot Spots
Andrea Zatkova ´,1Daniel Beltra ´n Valero de Bernabe ´,3,4Helena Pola ´kova ´,1Marek Zvarı ´k,2
Eva Fera ´kova ´,2Vladimir Bos ˇa ´k,5Vladimı ´r Fera ´k,2L’udovı ´t Ka ´dasi,1and
Santiago Rodrı ´guez de Co ´rdoba
3,4
1Institute of Molecular Physiology and Genetics and
Bratislava, Bratislava;
Inmunologı ´a, Centro de Investigaciones Biolo ´gicas, Consejo Superior de Investigaciones Cientı ´ficas, Madrid; and
Rheumatic Disease, Pies ˇt’any, Slovakia
2Department of Molecular Biology, Faculty of Natural Sciences, Comenius University
3Unidad de Patologı ´a Molecular and Unidad de Investigacio ´n, Fundacio ´n Jime ´nez Dı ´az, and
4Departamento de
5Research Institute of
Alkaptonuria (AKU) is an autosomal recessive disorder caused by the deficiency of homogentisate 1,2 dioxygenase
(HGO) activity. AKU shows a very low prevalence (1:100,000–250,000) in most ethnic groups. One notable
exception is in Slovakia, where the incidence of AKU rises to 1:19,000. This high incidence is difficult to explain
by a classical founder effect, because as many as 10 different AKU mutations have been identified in this relatively
small country. We have determined the allelic associations of 11 HGO intragenic polymorphisms for 44 AKU
chromosomes from 20 Slovak pedigrees. These data were compared to the HGO haplotype data available in our
laboratory for 180 AKU chromosomes from different European and non-European countries. The results show
that common European AKU chromosomes have had only a marginal contribution to the Slovak AKU gene pool.
Six of the ten Slovak AKU mutations, including the prevalent G152fs, G161R, G270R, and P370fs mutations,
most likely originated in Slovakia. Data available for 17 Slovak AKU pedigrees indicate that most of the AKU
chromosomes have their origins in a single very small region in the Carpathian mountains, in the northwestern
part of the country. Since all six Slovak AKU mutations are associated with HGO mutational hot spots, we suggest
that an increased mutation rate at the HGO gene is responsible for the clustering of AKU mutations in such a
small geographical region.
Alkaptonuria (AKU [MIM 203500]), the first human
disease to be interpreted as a recessive trait (Garrod
1902), is a rare disorder of the phenylalanine and ty-
rosine catabolic pathway caused by the deficiency of
homogentisate dioxygenase (HGO [E.C.1.13.11.5]) ac-
tivity (La Du et al. 1958). AKU patients are homozygous
or compound heterozygous for loss-of-function muta-
tions in HGO (Ferna ´ndez-Can ˜o ´n et al. 1996). As a con-
sequence of this defect, AKU patients cannot convert
homogentisate to maleylacetoacetate, which results in
homogentisic aciduria, ochronosis, and arthritis (La Du
Received July 19, 2000; accepted for publication September 13,
2000; electronically published October 2, 2000.
Address for correspondence and reprints: Dr. Santiago Rodrı ´guez
de Co ´rdoba, Departamento de Inmunologı ´a, CentrodeInvestigaciones
Biolo ´gicas, Consejo Superior de Investigaciones Cientı ´ficas, Vela ´zquez
144, 28006-Madrid, Spain. E-mail: SRdeCordoba@cib.csic.es
? 2000 by The American Society of Human Genetics. All rights reserved.
0002-9297/2000/6705-0032$02.00
et al. 1995). AKU presents a remarkable allelic hetero-
geneity. In a series of !100 unrelated patients frommany
different countries, 140 different AKU mutations have
been identified (Ferna ´ndez-Can ˜o ´n et al. 1996; Gehrig et
al. 1997; Beltra ´n-Valero de Bernabe ´ et al. 1998; Hi-
gashino et al. 1998; Ramos et al. 1998; Beltra ´n-Valero
de Bernabe ´ et al. 1999a, 1999b; Felbor et al. 1999;
Mu ¨ller et al. 1999; Walter et al. 1999; Porfirio et al.
2000; Zatkova ´ et al. 2000; authors’ unpublished data).
The most prevalent mutation in Europe (excluding the
Slovak AKU patients)is M368V,whichrepresents∼20%
of the AKU chromosomes. Similarly, V300G and P230S
each represent ∼5% of the European AKU chromo-
somes. In addition to the AKU mutations, 11 polymor-
phisms have been encountered within the human HGO
gene (Granadino et al. 1997; Beltra ´n-Valero de Bernabe ´
et al. 1998, 1999a; this report). The analysis of the hap-
lotypic association of these polymorphic markers in the
AKU chromosomes has shown that the three most dif-

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Am. J. Hum. Genet. 67:1333–1339, 2000
fused AKU mutations in Europe—M368V, V300G, and
P230S—are not recurrent mutations. Instead, they are
probably old mutations that were introduced in Europe
with the founder populations and have spread through-
out Western Europe with the different migrations (Bel-
tra ´n Valero de Bernabe ´ et al. 1998).
Slovakia is a country with a notable incidence of al-
kaptonuria (1:19,000) (Srsen et al. 1978). However, the
high frequency of AKU in this small geographical region
is difficult to explain. Recently, we and others have dem-
onstrated the existence of many different AKU muta-
tions in Slovakia, suggesting that several independent
founders have contributed to the AKU gene pool in this
geographical location (Gehring at al. 1997; Mu ¨ller et al.
1999; Zatkova ´ et al. 2000).
To get further insight into the history of the Slovak
AKU chromosomes and to provide an explanation for
the presence of multiple AKU mutations in Slovakia, we
have determined the allelic associations of 11 HGO in-
tragenic polymorphisms for 44 AKU Slovak chromo-
somes. Our sample of AKU Slovak chromosomes in-
cludes 29 chromosomes, corresponding to 13 AKU
pedigrees that have been reported elsewhere (Zatkova ´
et al. 2000), and 15 novel chromosomes from seven
patients with AKU who have not been reported. We
included in these studies all unrelated pedigrees from
Slovakia that we could obtain. Most of the samples were
obtained throughout the Research Institute of Rheu-
matic Diseases of Pies ˇt’any, whose activity covers all of
Slovakia. AKU mutations were identified in all 44 chro-
mosomes. As many as 10 different AKU mutations were
characterized in our sample: IVS1-1GrA, S47L, R58fs,
IVS5?1GrA, G152fs, G161R, P230S, G270R, V300G,
and P370fs. Interestingly, we identified no novel muta-
tions in the 15 new Slovak AKU chromosomes, sup-
porting the idea that our sample, including 44 AKU
chromosomes, is representative of the whole spectrum
of AKU mutations in Slovakia. As indicated, these AKU
mutations have been reported previously (Fernandez-
Can ˜o ´n et al. 1996; Gehrig et al. 1997; Beltra ´n-Valero
de Bernabe ´ et al. 1999a, 1999b; Mu ¨ller et al. 1999;
Porfirio et al. 2000; Zatkova ´ et al. 2000), and most of
them have been demonstrated to be loss-of-functionmu-
tations (Fernandez-Can ˜o ´n et al. 1996; Rodriguez et al.
2000; Titus et al. 2000; authors’ unpublished data).
The result of HGO-haplotype analysis in the Slovak
AKU chromosomes is summarized in figure 1. For com-
parison, figure 1 also includes the HGO haplotypes for
all the AKU chromosomes characterized thus far in our
laboratory from non-Slovak patients who carry theAKU
mutations found in the Slovak patients.
It is noticeable that M368V, the most prevalent AKU
mutation in Europe and a relatively frequent AKU mu-
tation in the neighboring countries, has not been en-
countered in the Slovak patients. Similarly, other AKU
mutations carried by patients from different countries
like P230S, V300G, R58fs, and IVS1-1GrA are rela-
tively infrequent in Slovakia. Nevertheless, the P230S,
V300G, R58fs, and IVS1-1GrA mutations in theSlovak
patients are found to be associated with the same HGO
haplotypes as previously described outside Slovakia, or
the differences (as for V300G) can be easily explained
by recombination (fig. 1). These data reinforce the con-
cept that IVS1-1GrA, R58fs, P230S, and V300G are
relatively old AKU mutations that spread in Europe with
different migrations. However, there is a low number of
AKU chromosomes carrying these mutations in oursam-
ple (7/44), which illustrates that these common Euro-
pean AKU chromosomes have had a marginal contri-
bution to the AKU gene pool in Slovakia.
The data depicted in figure 1 support the idea that
six of the AKU mutations found in our Slovak sample
probably originated in this geographical location. The
IVS5?1GrA mutation is particularlyinteresting,because
it has been found in three Slovak AKU chromosomes
associated with two different HGO haplotypes (fig. 1).
IVS5?1GrA is, therefore, a recurrent mutation in Slo-
vakia. In addition, this finding confirms that c.509?1 is
a hot spot of mutation in HGO. As noticed by Mu ¨ller et
al. (1999) and Zatkova ´ et al. (2000), this same HGO
nucleotide position has been described earlier to be mu-
tated to a T (IVS5?1GrT) in a Dutch patient with AKU
(Beltra ´n Valero de Bernabe ´ et al. 1998).
G270R is a prevalent mutation in Slovakia that has
also been found in an Italian patient (Porfirio et al.
2000). We have compared the HGO haplotypes asso-
ciated with this mutation in the Italian and Slovak pa-
tients and found that they differ at both the 3?and the
5?ends of the HGO gene, suggesting two independent
origins for this mutation (fig. 1).
A Slovak origin for the G152fs, G161R, and P370fs
mutations is supported both by their high prevalence in
Slovakia and by the fact that they are found almost
exclusively in this country. G152fs and G161R occa-
sionally have been found outside Slovakia, but we could
not exclude a Slovak origin for these few patients with
AKU. The HGO-haplotype analysis also provides evi-
dence of recombination in the AKU chromosomes car-
rying the G161R and P370fs mutations, which indicates
that some of the Slovak mutations may be relatively old
mutations (fig. 1). The S47L mutation is a very rareAKU
mutation that has been found only in one Slovak patient.
The HGO-A, HGO-B, HGO-C, HGO-D, and HGO-
E haplogroups were initially defined considering only 7
of the 11 HGO polymorphic sites described here:
IVS2?35, c.407, HGO-3, HGO-1, IVS5?25, IVS6?46,
and HGO-2 (Beltra ´n Valero de Bernabe ´ et al. 1998).
Analysis of these HGO haplogroups in 90 individuals
from the Slovak population without AKU shows a dis-

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Figure 1
morphisms for each of the 44 AKU Slovak chromosomes included in this study. The HGO polymorphic loci, ordered from 5?to 3?, are IVS2?35,
IVS2-218, IVS3-112, Ex4 (c407), IVS4?31, HGO-3, HGO-1, IVS5?25, IVS6?46, IVS11?18, and HGO-2. HGO-1, HGO-2, and HGO-3
are (CA)n or (CT)n dinucleotide repeats (Granadino et al. 1997; Beltran Valero de Bernabe et al. 1998). All other polymorphisms are diallelic
SNPs (Beltran Valero de Bernabe et al. 1998; 1999a). AKU chromosomes are grouped by mutations. Each mutation group also includes the
chromosomes described thus far outside Slovakia that carry the same AKU mutation. The chromosomes are identified by the pedigree code
number, followed by a, b, c, or d (a and b indicates that the patient is an HGO homozygote). A thick vertical bar indicates the position, in
the HGO haplotype, of each AKU mutation. A grey color code is used to identify the different HGO haplotypes. In very few instances, there
was no information for the segregation of the alleles and both alleles were included in the haplotype. PR p present report.
HGO haplotypes associated with the AKU mutations. The figure shows the allelic associations of 11 HGO intragenic poly-
tribution that is not significantly different from that de-
scribed earlier for other populations (table 1).
Analysis of the full HGO haplotypes (including the
11 polymorphic sites) in this relatively small sample of
the unaffected Slovak populations demonstrated the
presence of HGO haplotypes that were identicaltothose
associated with the G161R, G270R, G152fs, and
IVS5?1GrA mutations. Further analysis of these un-
affected individuals demonstrated that none of the in-
dividuals who carry the HGO haplotype associatedwith
the IVS5?1GrA mutation (three individuals) or the
G152fs mutation (two individuals) are AKU heterozy-
gotes. Interestingly, however, one of the two control in-
dividuals who carry the HGO chromosomes associated
with the G161R mutation and one of the two who carry
the HGO chromosomes associated with the G270R mu-
tation are also carriers of the G161R or G270R AKU
mutation, respectively. These data further illustrate the
extraordinary frequency (1/90) of AKU alleles in the
unaffected Slovak population and demonstrate a coex-
istence of identical HGO haplotypes (including the 11
polymorphic sites) with and without AKU mutations in
this population.
To get an insight into the history of the different AKU
mutations in Slovakia, we have investigated the geo-
graphical origins of the maternal and paternal grand-
parents of the AKU patients. This information wasavail-
able for 17 AKU pedigrees, which allowed us to deter-
mine a geographical origin for 37 AKU chromosomes,
including eight different mutations. Figure 2 summarizes
these data and illustrates an extraordinary clustering of
the mutations in a very small geographical area in the

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Am. J. Hum. Genet. 67:1333–1339, 2000
Table 1
Most-Representative HGO Haplogroups in the Slovak Population without AKU
HAPLOGROUP
APPROXIMATE
FREQUENCY
ALLELES FOUND TO BE ASSOCIATED WITH HAPLOGROUP
IVS2?35A/T c407A/T
HGO-3a
HGO-1IVS5?25T/CIVS6?46A/C
HGO-2a
HGO-A .57A T (50%)
A (50%)
193 (48%)
189 (14%)
195 (14%)
197 (14%)
161TC 181 (16%)
187 (36%)
183 (12%)
189 (9%)
191 (8%)
177 (35%)
175 (30%)
HGO-B .12AT 189 (40%)
197 (20%)
193 (20%)
191 (89%)
191 (91%)
189 (50%)
191 (30%)
193 (20%)
161 TA
HGO-C
HGO-D
HGO-E
.11
.14
.06
T
T
A
T
T
T
161
161
163
T
T
C
A
C
A
179 (61%)
…b
187 (60%)
185 (20%)
183 (20%)
aHGO-2 and HGO-3 alleles that are found to be predominantly associated with each of the HGO haplogroups.
bNo specific allele was found to be associated with this haplogroup.
Carpathian mountains, the so-called “Kysuce” region,
around the city of Cadca. This remarkable situation was
first described by Mu ¨ller et al. (1999), who identified,
in this isolated Slovak region, using anindependentAKU
population sample, seven families carrying five different
AKU mutations.
The only information available for the Slovak AKU
pedigrees carrying the common European P230S and
V300G mutations is that they live in the city of Zilina.
However, Mu ¨ller et al. (1999) found these mutations
only in the central and southern parts of Slovakia.
The Kysuce region in Slovakia, where most of the
AKU mutations concentrate, is believed to have be pop-
ulated in the 14th and 15th centuries by Valachian im-
migrants. Valachians were nomadic tribes of Romanian
origin who came to Slovakia from the Balkan countries
through western Ukraine. Looking for new pastures,
they moved to the north throughout the Carpathian
mountains. Among other places, they settled in the val-
leys of river Kysuca, where they remained isolated until
the end of World War II (Srsen 1984). Nowadays, there
are populations with Valachian origins in other Euro-
pean countries, such as Romania, Moldavia, Greece,
Bulgaria, Hungary, and Albania. However, there is no
indication of a high incidence of AKU in these popu-
lations, suggesting that the AKU mutations were not
introduced into the Kysuce region by this colonization.
The presence of multiple mutations in a single gene
in a population living in a small geographical region has
been described elsewhere for other disorders, like Hurler
syndrome and metachromatic leukodystrophy (MDL) in
Lower Galilee (Bach et al. 1993; Heinisch et al. 1995),
and limb-girdle muscular dystrophies (LGMDs) on La
Reunion island (Richard et al. 1995). However, in the
case of Kysuce, the number of different mutations is
remarkably high.
It is difficult to explain the presence of multiple mu-
tations in a specific gene that are restricted to a small
geographical area. One hypothesis about the extraor-
dinary allelic heterogeneity of AKU in the Kysuce region
involves different founders who immigrated into Slo-
vakia at different times in history and settled close to
each other in this region of the Carpathian mountains.
It is, however, difficult to imagine how genetic drift or
selective pressures (including social pressures) could
have driven families with affected children to migrate
together and settle in the Kysuce region. As indicated
above, the most prevalent and ancient AKU mutations
in Europe (M368V, V300G, and P230S) seem to be ab-
sent from the critical Kysuce region, which, in turn, clus-
ters a number of AKU mutations found almost exclu-
sively in Slovakia. This peculiar distribution of the AKU
mutations and the uniqueness of the Kysuce region in
Slovakia, regarding the clustering of AKU mutations, fit
best with the idea that a significant number of the Slovak
AKU mutations originated in theKysuceregionandthat,
during recent times, emigrants from the Kysuce region
have disseminated these AKU mutations throughoutSlo-
vakia. In this regard, it is noticeable that the six AKU
mutations that we believe have originated in this region
are associated with HGO mutational hot spots.
IVS5?1GrA was mentioned above and represents a
clear HGO mutational hot spot. P370fs, G161R,
G270R, and G152fs are mutations that involve CCC (or
GGG) triplets, a sequence motif that has been shown to
be hypermutable in the HGO gene (Beltra ´n Valero de
Bernabe ´ et al. 1999a). Finally, S47L originates from a
CrT change at nucleotide position c.307, within a CpG

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Figure 2
AKU chromosomes included in this report. No family data relative to geographical origins were available for the remaining 7 AKU chromosomes.
The code used for the mutations is depicted in the right inset.
Geographical distribution of the Slovak AKU mutations The figure shows the geographical locations for 37 of the 44 Slovak
dinucleotide that is predicted to be a mutational hot spot
by the MUTPRED program (Cooper and Krawczak
1990).
Both the molecular mechanisms that could be re-
sponsible for the hypermutation process at CCC (or
GGG) triplets(orat nucleotidec.509?1)andthereasons
why this process appears to be restricted to the HGO
gene are unknown. High mutation rate could resultfrom
both a sequence motif prone to mutation and some in-
terference with the nucleotide-repair machinery. Simi-
larly, differences between genes for the occurrence of
mutations at specific short sequence motifs are perhaps
a consequence of structural (sequence modification,
chromatin organization, etc.) and functional (transcrip-
tion rate, etc.) features of the individual genes. The ob-
servation that several AKU mutations associated with
this mutational hot spot have originated in a small ge-
ographical area in Slovakia may offer an opportunity to
analyze these possibilities. In this regard, it would be
interesting to determinewhetherparticulardietsorliving
conditions may have exposed the inhabitants in this re-
gion of Slovakia to chemical or physical agents with the
ability to induce specific mutations in specific human
genes.
In conclusion, in this report, we provide new data that
allow us to formulate novel hypotheses to explain the
incidence of AKU in Slovakia. We show that various
factors have probably contributed to the AKU gene pool
in Slovakia. Some mutations—such as P230S, V300G,
R58fs, and IVS1-1GrA—are shared by different pop-
ulations and were probably introduced to Slovakia with
the founder populations that spread throughout Europe.
However, these mutations represent only 16% of the
Slovak AKU chromosomes. The most prevalent muta-
tions in Slovakia (G152fs, G161R, G270R, and P370fs)
are relatively old mutations that most likely originated
at a single and very small geographical location, the
Kysuce region, in the northern part of Slovakia. These
mutations probably spread from this region to other
regions of Slovakia, as suggested by the radiation pat-
terns observed for two of these mutations, G161R and
G152fs (see fig. 2 and also Mu ¨ller et al. [1999]). Inter-
estingly, the G152fs, G161R, G270R, and P370fs mu-
tations all involve CCC (or GGG) triplets, a sequence

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Am. J. Hum. Genet. 67:1333–1339, 2000
motif that behaves as a mutational hot spot in the HGO
gene (Beltra ´n Valero de Bernabe ´ et al. 1999a). On the
basis of these results, we postulate that the remarkable
allelic heterogeneity of AKU in Slovakia was a conse-
quence of an increased mutation rate at the HGO gene
in the Kysuce region. This increased mutation rate in-
volved different mutational hot spots. Subsequent ge-
netic drift and isolation preserved and increased the fre-
quency of these mutations and led to the high frequency
of alkaptonuria in Slovakia.
Acknowledgments
We thank the families with AKU and all the clinicians, par-
ticularly Drs. F. Cisarı ´k, M. Lukae `ovie `, N. Mis ˇovicova ´, J. Krs ˇi-
akova ´, A. Kanabova ´, and Prof. J. Rovensky ´, D.Sc., for their
collaboration and donation of blood (DNA) samples. We
would also like to thank I. Szomolayova ´ and L. Gulliksen for
their excellent technical assistance and contribution to this
work. This research was supported by the Fundacio ´n Jose An-
tonio de Castro, the Spanish Comisio ´n Interministerial de
Ciencia y Tecnologı ´a (SAF99/0013), and the Comunidad de
Madrid (08.6/0015/1997). In addition, this study is based on
work supported by the Fundacio ´n Conchita Ra ´bago de
Jime ´nez Dı ´az, under a grant awarded to D.B.-V.de B., and
partially by HESP grant number G/004/00/61010, awarded to
M.Z. by the Open Society Foundation, Bratislava.
Electronic-Database Information
Accession numbers and URLs for data in this article are as
follows:
AKU database, http://www.cib.csic.es/˜akudb/index.htm (for
published and unpublished data of mutations and poly-
morphisms in the HGO gene)
Entrez, http://www.ncbi.nlm.nih.gov/Entrez (for genomic se-
quences of HGO and its transcript; accession numbers
AF000573 and AF045167, respectively)
Online Mendelian Inheritance in Man (OMIM), http://www
.ncbi.nlm.nih.gov/Omim (for AKU [MIM 203500])
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