In Slovakia, within the rather relatively small population of 5.5 million, 208 patients have been registered (Sršeň et al. 2002) and a total of 12 different HGD mutations have been established, revealing a remarkable allele heterogeneity of AKU in this country. An allelic association was performed for 11 HGD intragenic polymorphisms in a total of 69 AKU chromosomes from 32 Slovak pedigrees. These were then compared to the HGD haplotypes of all AKU chromosomes carrying identical mutations characterized thus far in non-Slovak patients in order to study the possible origin of these mutations (Zat´ková 2011). Based on the analysis and comparison of haplotypes, two groups of HGD mutations were observed in Slovakia. To the first group belong mutations P230S, V300G, S59fs (R58fs), M368V and IVS1-1 G > A, which account for 17.3 % of the Slovak AKU chromosomes and thus provide a marginal contribution to the AKU gene pool in this country. The most frequent European mutation, M368V, is present in one copy in only two unrelated Slovak families. Mutations of this group are shared by different populations and have most likely been introduced into Slovakia by the founder populations that spread throughout Europe (Zat´ková et al. 2000a, b, c). The second group consists of the remaining seven mutations established in 82.7 % of Slovak patients. These include the most prevalent G161R (44.2 %), D153fs (G152fs) (14.4 %), H371fs (P370fs) (11.5 %) and G270R (7.7 %), as well as IVS5 + 1 G > A present on three AKU chromosomes and the S47L and E178G mutations observed each in only one patient. It is likely that mutations from this second group originated in Slovakia. The distribution of the identified mutations within Slovak territory is also interesting. As previously reported, examination of the geographical origin of Slovak AKU mutations shows remarkable clustering in a small area in northwest Slovakia, with these mutations most likely originating in this area and spreading into other regions after the breakdown of genetic isolates in the 1950s (Zat´ková et al. 2000a, b, c). As the combined sequence and haplotype analysis shows, 7 of the 12 AKU mutations (58.3 %) that most likely originated in Slovakia are associated with hypermutated sequences in the HGD, whilst worldwide it is 40/115 (34.8 %). Therefore, it is possible that an increased mutation rate in the HGD gene in a small geographical region is responsible for the high genetic heterogeneity in Slovak AKU (Zat´ková et al. 2000a, b, c). However, it remains unclear which mechanism acted specifically on the HGD gene to increase its mutation rate, since similar targets are also present in other genes without evident elevated gene frequency in Slovakia (Sršeň et al. 2002; Zat´ková et al. 2000a, b, c). The increased number of mutations could also be the result of random accumulation of mutations in the region. It has been suggested that the Valachian colonization during the fourteenth to seventeenth centuries may also have played a role in the increased prevalence of AKU in Slovakia (Sršeň et al. 2002; Zat´ková et al. 2000a, b, c). The preservation of the most prevalent AKU variants, which either arose in Slovakia or were brought there, may be the result of a founder effect and genetic drift, due to the geographic isolation of villages in northwest Slovakia.

AKU belongs to rare diseases from the point of population genetics. Only slightly over 1,000 cases have been reported in the literature. The worldwide prevalence of AKU is not precisely known; it is estimated to be 1:250,000–1,000,000 (Beighton et al. 1993). However, it has got wide ethnic and geographic distribution. Its occurrence has been reported in various populations in Europe, America and Asia, i.e. this disease is not restricted to a certain race, but it affects individuals of various ethnic groups. It is interesting that AKU occurrence was not observed in the Gypsy ethnic group in Slovakia. Findings of typical ochronotic spondylosis in Egyptian mummies dated about 1500 B.C. suggest its occurrence in the ancient past (Beighton et al. 1993). The highest worldwide incidence of AKU is found in the Dominican Republic and Slovakia. For example, overall 149 patients were registered in Slovakia in the 1980s. A 6-year screening of the newborn population in Slovakia (417,122 newborns who survived the first 4 weeks of life) revealed about 1:19,000 incidence (Sršeň 1984). In certain regions of Slovakia, the disease incidence was up to ten times higher. These regions developed for a long time (until World War II) as genetic isolates, e.g. Kysuce, Orava and surroundings of Trenčín and Horehronie – the inbreeding coefficient (F) ranged from 0.003 to 0.029 (Sršeň 1984). The higher AKU incidence in these regions is probably a consequence of their long-term geographic isolation and endogamy, in addition to such genetic factors like genetic drift and founder effect. Break-up of isolates after World War II resulted in the gradual decrease of AKU incidence in Slovakia. On the basis of genetic population analyses, the frequency of gene/allele for AKU in Slovakia was estimated to be 0.7 % – q = 0.007 (Sršeň 1984). This value is relatively high, but in contrast to AKU frequency, it will not be significantly affected by break-up of isolates. On its basis, it is possible to perform certain genetic estimates that are not accurate with regard to several factors, which have to be taken into account in population analyses. However, they enable us to obtain several data of orientation value that can be useful from a practical point of view. On the basis of these estimates, there could be more than 250 patients with AKU in the whole current population of Slovakia (about 5.5 million citizens). The frequency of heterozygotes, i.e. healthy carriers of the pathological allele, would be approximately 14 persons per 1,000, and two matrimonies out of 10,000 would carry the risk of 25 % of two healthy heterozygotes having children with AKU. It has to be emphasized that in the case of consanguineous marriages or marriage of the people who come from the above-mentioned endogamous locations, the risk of AKU incidence in children is even higher. These data demonstrate that AKU problems and of course subsequent ochronosis and ochronotic arthropathy are topical in the Slovak population even nowadays and attention has to be paid to this disease.

With regard to the aforementioned frequent incidence of AKU in the Slovak population, it has been the subject of long-term research. Four institutions in Slovakia were devoted to its study – National Institute of Rheumatic Diseases in Piestany, Institute of Clinical Genetics of Jessenius Medical Faculty of Comenius University in Martin, Institute of Molecular Physiology and Genetics of Slovak Academy of Sciences in Bratislava and Faculty of Natural Sciences of Comenius University in Bratislava. The following years are important from the historical point of view:

Advances in the field of AKU molecular genetics contributed not only to a better understanding of the disease etiopathogenesis, but they were also significant from a practical point of view as they enabled disease diagnostics at DNA (gene) level which gives a high-quality definitive diagnosis. DNA analysis can be used for identifying healthy carriers of the pathological allele (heterozygotes), which was not possible by biochemical methods. This knowledge can be used in genetic counselling. Knowledge of the molecular-genetic basis of AKU is very promising for the development of new approaches to the therapy of this disease which is currently aimed at symptomatic treatment of some complications and is not always effective. Two options of causal therapy are possibilities – substitute administration of HGD enzyme and gene therapy at the level of the somatic cell. Direct administration of the enzyme faces several problems, especially its localization in the liver, but also the route, the dose and the period and interval of administration. The most convenient option would be the application of the so-called recombinant enzyme. An example of such replacement enzymatic therapy is Gaucher’s disease. Somatic gene therapy currently moves from animal studies to clinical trials in humans, e.g. in phenylketonuria. The normal functional gene is given to the patients using a suitable vector. According to current knowledge and experience with gene therapy, it is sufficient to achieve 5 % expressivity of the gene in order to yield its curative effect. Such an approach is supported by the recent results of a liver transplantation in an AKU patient in whom the progression of clinical and radiological changes was stopped, because hepatocytes of the transplanted liver had a functional HGD gene (Kobak et al. 2005). Gene therapy is a great hope for AKU patients because we still cannot treat this disease, which often causes disability and considerable suffering. Several other treatment strategies have also been suggested for AKU including nitisinone, the triketone herbicide, which inhibits the 4-hydroxyphenylpyruvate dioxygenase enzyme that produces HGA (Suwannarat et al. 2005).