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Alkaptonuria in the Dominican Republic: identification of the founder AKU mutation and further evidence of mutation hot spots in the HGO gene
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1. E Goicoechea de Jorge1,3,
2. I Lorda2,
3. M E Gallardo1,
4. B Pérez1,3,
5. C Peréz de Ferrán4,
6. H Mendoza4,
7. S Rodríguez de Córdoba1,3
1. 1Unidad de Patología Molecular, Fundación Jiménez Díaz, Av Reyes Católicos 2, 28040 Madrid, Spain
2. 2Servicio de Genética, Fundación Jiménez Díaz, Av Reyes Católicos 2, 28040 Madrid, Spain
3. 3Departamento de Inmunología, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Velázquez 144, 28006 Madrid, Spain
4. 4Centro Nacional de Investigación en Salud Materno Infantil, Hospital de Niños Robert Reid Cabral, Abraham Lincoln 2, Santo Domingo, Dominican Republic
1. Correspondence to:  Dr S Rodríguez de Córdoba, Departamento de Inmunología. Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Velázquez 144, 28006-Madrid, Spain;  SRdeCordoba{at}cib.csic.es

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Alkaptonuria (AKU, MIM 203500), the first human disease to be recognised as a recessive trait and Archibald Garrod's prototype “inborn error of metabolism”,1,2 is a rare disorder of the phenylalanine and tyrosine catabolic pathway caused by the deficiency of homogentisate dioxygenase (HGO, EC 1.13.11.5) activity.3 AKU patients are homozygous, or compound heterozygous, for loss of function mutations in HGO.4 As a consequence of this defect, AKU patients cannot convert homogentisate to maleylacetoacetate, which results in homogentisic aciduria, ochronosis, and arthritis.5 AKU shows remarkable allelic heterogeneity. More than 40 different AKU mutations have been identified in a total of fewer than 100 unrelated patients from many different countries. In addition to the AKU mutations, 19 polymorphisms have been encountered within the human HGO gene (for a complete description of the HGO mutations and polymorphisms see the AKU database (http://www.cib.csic.es/~akudb/index.htm)). The analysis of the haplotype association of polymorphisms in the AKU chromosomes has been very useful for the identification of the different AKU alleles and for tracing their migration during recent human history. In this regard, it has been shown that the three most widespread AKU mutation in Europe, M368V, V300G, and P230S (representing 20%, 5%, and 5% of European AKU chromosomes, respectively) are not recurrent mutations. Instead they are probably old mutations that were introduced into Europe with the founder populations and have spread throughout western Europe with the different migrations.6 Analysis of the HGO mutations and polymorphisms has also shown that the GGG sequence motif (or its reverse complement CCC) is a mutational hot spot in the HGO gene.7

AKU has a very low prevalence (1:100 000-250 000) in most populations. However, in certain areas, such as the Dominican Republic and Slovakia, the incidence of alkaptonuria is unusually high.8,9 In Slovakia, AKU has an approximate incidence of 1:19 000 and shows unexpected allelic heterogeneity. As many as 10 different AKU mutations have been identified in AKU Slovak patients, suggesting that several independent founders have contributed to the AKU gene pool in this geographical location.10–12 Recently, we provided evidence indicating that the most prevalent Slovak mutations (G152fs, G161R, G270R, and P370fs) most likely originated in Slovakia at a single and very small geographical location, the Kysuce region, in the northern part of the country.11 Our data also suggested that increased mutation rates in HGO, associated with GGG triplets and other mutational hot spots, were probably involved in the origin of these Slovak mutations.

No molecular data are yet available from the Dominican AKU patients. However, early epidemiological data suggest that the high incidence of AKU in the Dominican Republic could be the consequence of a classical founder effect. In these early studies, as many as 47 AKU cases were reported occurring in eight highly inter-related kindreds in a mixed Spanish-Indian group living in rural Santo Domingo.8

To determine whether the high incidence of AKU in the Dominican Republic is the consequence of a founder effect (as suggested by the epidemiological data) or an increased mutation rate in the HGO gene (as seems to have been the case in Slovakia), we have performed HGO mutation/polymorphism analyses in eight unrelated Dominican AKU patients and their relatives. These patients are all from the same geographical area as the eight kindreds described by Milch8 in 1960 and we have confirmed that at least one of the Dominican AKU patients included in our study belongs to one of those kindreds. This patient and the common geographical origin allow us to link the present molecular studies with Milch's epidemiological data.8

## MATERIAL AND METHODS

DNA from patients and their relatives was extracted from buccal mucosa cells collected with MasterAmp buccal brushes (Epicentre Technologies, USA) following informed consent. Mutation and polymorphism analyses were performed as previously described.6 Briefly, the 14 exons of the HGO gene were amplified from genomic DNA using specific primers derived from the 5 and 3 intronic sequences and the corresponding PCR products were purified using Wizard PCR Preps DNA Purification System (Promega, USA). Direct sequencing of the PCR products was performed with a dye terminator cycle sequencing kit (Applied Biosystems, USA) and resolved in an ABI PRISM 3700 automatic DNA sequencer. Analysis of polymorphisms at the HGO-1 (D3S4496), HGO-2 (D3S4497), and HGO-3 (D3S4556) microsatellites was performed by PCR using total human genomic DNA. The HGO intragenic dimorphic markers IVS3-112T/C, c407T/A, IVS4+31A/G, IVS5+25T/C, IVS6+46C/A, and IVS11+18A/G were characterised in the same PCR fragments that were sequenced during the search for AKU mutations.

## RESULTS

AKU mutations were identified in all 16 Dominican AKU chromosomes. Seven of the eight AKU patients are homozygous for a c.527T→G substitution in exon 6 of the HGO gene, resulting in a missense mutation (C120W), which replaces a cysteine in position 120 with a tryptophan. The remaining Dominican AKU patient is homozygous for the G270R (c.975G→A) missense mutation. G270R has been described previously as a prevalent AKU mutation in Slovakia.11,13

We used the recently discovered HGO crystal structure14 to determine the consequences of the C120W and G270R mutations. C120W is expected to have a strong disruptive effect on the HGO active site, whereas G270R interferes with the formation of the HGO hexamer. Therefore, both mutations have structural consequences that should impair HGO enzyme activity.

The haplotype associations between the AKU mutations and nine polymorphic sites within the HGO gene were established for the 16 Dominican AKU chromosomes. Fig 1 shows that in all 14 C120W chromosomes the AKU mutation is associated with identical polymorphisms and that, therefore, these seven Dominican AKU patients should share a common ancestor from whom they have inherited the AKU mutation. Moreover, since one of the seven AKU patients carrying the C120W mutation belongs to the very large AKU pedigree described in 1960,8 it is reasonable to expect that C120W is the founding mutation common to most AKU patients in the Dominican Republic. It is interesting in this regard that the only other AKU patient previously identified carrying this mutation was a 75 year old Dominican male living in the USA.16 However, no HGO haplotype data are available from this patient. Whether the C120W mutation originated in the Dominican Republic or was introduced there by immigrants from other geographical regions is at present unknown.

Figure 1

HGO haplotypes associated with the Dominican AKU mutations. The figure shows the allelic associations of nine HGO intragenic polymorphisms for each of the 16 Dominican AKU chromosomes included in this study. The HGO polymorphic loci ordered from 5 to 3 are indicated at the top. HGO-1, HGO-2, and HGO-3 are (CA)n or (CT)n dinucleotide repeats. All other polymorphisms are diallelic SNPs. AKU chromosomes are grouped by mutation. In the G270R mutation group, we have also included the chromosomes described in Italy and Slovakia that carry the same AKU mutation. The position of the AKU mutations within the HGO haplotype is indicated with a vertical bar. The chromosomes are identified by the pedigree code number. PR, present report.

One of the eight Dominican AKU patients is homozygous for the G270R mutation. As indicated above, the G270R mutation is one of the most prevalent AKU mutations in Slovakia. We have previously indicated that the G270R mutation originated in Slovakia associated with a GGG mutation hot spot.11,13 Interestingly, the Slovak and the Dominican G270R mutations are associated with two completely different HGO haplotypes (fig 1), illustrating that they have originated through independent mutational events.

### Key points

• The aim of this study was to determine whether the high incidence of AKU in the Dominican Republic is the consequence of a founder effect or an increased mutation rate in the HGO gene, as seems to have been the case in Slovakia, the only other known location with a high incidence of AKU.

• We performed HGO mutation analysis in eight unrelated Dominican AKU patients and their relatives and characterised the 16 HGO haplotypes by analysing nine intragenic polymorphisms.

• AKU mutations were identified in all 16 Dominican AKU chromosomes. Seven of the eight AKU patients are homozygous for a c.527T→G substitution in exon 6 of the HGO gene, resulting in a missense mutation (C120W), which replaces a cysteine in position 120 with a tryptophan. The remaining Dominican AKU patient is homozygous for the G270R (c.975G→A) missense mutation.

• This study provides molecular evidence that the increased incidence of AKU in the Dominican Republic is the consequence of a classical founder effect and that C120W is the founder mutation. We have also identified a Dominican AKU patient homozygous for the G270R mutation and have shown that G270R, one of the prototype Slovak AKU mutations, is a recurrent mutation in the Dominican Republic. This finding reinforces our previous conclusion that the GGG sequence motif, or its reverse complement CCC, is an important mutational hot spot in the HGO gene.

In conclusion, HGO mutation analysis in 16 AKU chromosomes from eight unrelated patients from the Dominican Republic has shown two AKU mutations, C120W and G270R. C120W is the most prevalent mutation (87% of the AKU chromosomes tested) and most likely the mutation responsible for AKU in the 47 patients described in Milch's epidemiological report published in 1960.8 These data provide molecular evidence that the increased incidence of AKU in the Dominican Republic is the consequence of a classical founder effect and that C120W is the founder mutation. The identification of a patient carrying a homozygous G270R mutation suggests the existence of additional AKU founders at specific locations within the Dominican Republic. We have shown here that G270R, one of the prototype Slovak AKU mutations, is a recurrent mutation in the Dominican Republic. This finding reinforces our previous conclusion7 that the GGG sequence motif, or its reverse complement CCC, is an important mutational hot spot in the HGO gene.

## Acknowledgments

We thank the families with AKU for their collaboration and donation of blood (DNA) samples. We would also like to thank L Gulliksen for her contribution to this work. This research was supported by the Fundación José Antonio de Castro, the Spanish Comisión Interministerial de Ciencia y Tecnología (SAF99/0013), and the Comunidad de Madrid (08.6/0015/1997).

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