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Editor—Alkaptonuria (AKU, McKusick No 203500), a rare autosomal recessive disorder (1:250 000),1 is a classical example of a specific biochemical lesion leading to degenerative disease. As a result of deficiency of homogentisic acid 1,2-dioxygenase activity (HGO, E.C. 1.13.11.5), AKU patients are unable to degrade homogentisic acid (HGA), an intermediary metabolite in phenylalanine and tyrosine catabolism.2 Accumulated HGA is excreted into the urine in large amounts, which darkens on standing. Over the years, benzoquinone acetic acid, an oxidation product of HGA, is deposited in connective tissues, causing their pigmentation (ochronosis), which leads to painful and disabling arthropathy of the large joints and spine (ochronotic arthropathy).
AKU was the first disease interpreted in terms of Mendelian inheritance.3 The HGO gene in humans is located on chromosome 3q21-23.1 4 5Fernandez-Canon et al 5 cloned the human HGO gene and by identifying the first loss of function mutations also provided formal proof that AKU results from a defect in this gene. So far, 24 different mutations have been identified in the HGO gene in patients from various populations.5-10
Notable exceptions to the low prevalence of AKU in all ethnic groups studied are the Dominican Republic and Slovakia (1:19 000).11 12 Founder effects as the consequence of genetic isolation have been postulated to explain this observation. Here, we present results of mutation screening of theHGO gene in 32 AKU chromosomes carried by 17 Slovak AKU patients (in two families, one chromosome was shared by two patients from different generations). All 14 exons of theHGO gene were amplified from genomic DNA, using PCR primers and conditions as described by Fernandez-Canonet al. 5 PCR products were analysed for the presence of mutations by non-radioactive single strand conformation polymorphism analysis (SSCP).13 DNA was visualised by silver staining essentially as described by Budowleet al. 14 Fragments showing SSCP shifts were sequenced directly using the dye terminator cycle sequencing kit (Perkin Elmer) with Taq FS DNA polymerase. Sequences were resolved on an ABI-310 Automatic Analyser.
In our patients, we identified nine different mutations (tables 1 and2). Four of them were novel mutations, two missense (S47L, G270R), a frameshift (P370fs), and a splice site mutation (IVS5+1G→A), increasing the total number of known AKU causing nucleotide changes within the HGO gene to 28. The remaining five mutations have been described previously: G161R and G152fs,6 P230S and V300G,5 7 and IVS1-1G→A.9
List and frequencies of the mutations identified in 32 AKU chromosomes from Slovak patients. Positions of nucleotide changes are related to the transcription start site as described in Granadino et al.15 (Human HGO transcript: AF O45167; the ATG initiation codon is located at position c168)
Genotypes of all analysed AKU patients from 15 families indicating identified disease causing mutations within the HGO gene
Novel mutation S47L is caused by a transition C→T at the second position of codon 47 (fig 1A). This transition abolishes a restriction site for MboI in exon 3 PCR fragments. The presence of the S47L mutation in our patient was confirmed byMboI digestion (fig 2A).
Part of the direct sequencing of exons 3 (A), 5 (B), 11 (C), and 13 (D) in patients heterozygous for mutations S47L, IVS5+1G→A, G270R, and P370fs, respectively (ABI 310, Perkin Elmer). In the case of exons 3, 5, and 11, reverse primers were used.
Detection of the (A) IVS1-1G→A, S47L (c307C→T) and (B) G270R (c975G→A) mutations by RsaI, MboI, and EcoNI restriction analysis, respectively. Mutations IVS1-1G→A and S47L abolish restriction sites for corresponding enzymes, while G270R creates a novel site for EcoNI. The figure also shows segregation of mutations IVS1-1G→A in family ALK6 (A) and G270R in family ALK4 (B). In both families, the fathers and affected daughters are heterozygous for screened mutations ((A) lanes 1 and 4, (B) lanes 2 and 4, as well as the patient in (A) lane 6). In (A) lanes 2, 3, 7, and 8 and (B) lanes 3 and 5 are subjects without corresponding mutations. In (A) lane 5 and (B) lane 1 is a 100 bp ladder.
Mutation G270R is caused by transition G→A at the first position of codon 270, which creates a novel EcoNI restriction site (fig 1C). Therefore, its presence in our patients was confirmed by restriction digestion of exon 11 PCR fragments withEcoNI (fig 2B). Glycine at position 270, affected by this mutation, is conserved in man, mouse, andAspergillus nidulans (fig 3).
Comparison of primary structure of homogentisate-1,2-dioxygenase protein from man (HGO,AF000573 15), mouse (MHGO, U58988 16), and Aspergillus nidulans (HMGA, U30797 17) using ClustalX 1.3b. Positions conserved in all three organisms are indicated by (*). Arrows mark sites of identified missense mutations, novel mutations are shown in bold.
Splice site mutation IVS5+1G→A affects the donor splice sites of intron 5 (fig 1B). Interestingly, Beltran-Valero de Bernabéet al 7 identified in one patient from Holland a transversion G→T affecting the same position of intron 5 as our mutation IVS5+1G→A. This mutation, however, was not identified in our patients.
Mutation IVS1-1G→A9 abolishes restriction sites forRsaI, so the presence of this mutation on one AKU chromosome was confirmed by restriction analysis of the corresponding PCR fragment with this enzyme (fig 2A).
A novel P370fs frameshift mutation, caused by a single base insertion c1273insC (fig 1D), brings about a premature translation stop four codons downstream and subsequent shortening of translated HGO protein from 445 to 373 amino acids.
The novel mutations were not identified in any of the 50 healthy controls, supporting the evidence that they are disease causing mutations, rather than polymorphisms.
Segregation of all mutations with AKU was confirmed in all families, except for the S47L mutation, where no DNA from family members was available (fig 4). However, serine at position 47 of the HGO protein molecule is conserved in man and mouse (fig 3). InAspergillus nidulans, threonine is found at this site, which, as well as serine, belongs to the group of hydrophilic amino acids with uncharged polar side chains that are usually on the outside of the protein. Conversely, leucine, which is introduced into the HGO protein by transition c307C→T, is an amino acid with non-polar side chains that tend to cluster together on the inside of proteins. This indicates that substitution S47L may influence the HGO protein conformation and therefore also affect its function.
Non-radioactive SSCP analysis of exon 13 indicating segregation of mutation P370fs in family ALK5. Arrows mark the SSCP shifts corresponding to this mutation. The presence of the mutation in heterozygous state is indicated by (*). Patients were also heterozygous for mutation G270R (exon 11) (table 2).
Recently, Beltran-Valero de Bernabé et al 9 provided the evidence that the CCC triplet or its inverted complement (GGG) are mutational hotspots in theHGO gene, because 34.5% (10/29) ofHGO nucleotide changes identified so far involve these sequence motifs. Data shown in our report further support their finding, since 55.5% (5/9) of the mutations identified in our patients lie within or are adjacent to these triplets. Taking into account the novel mutations found in Slovak patients and one identified by Felbor et al,10 the total number of HGO nucleotide variations involving the CCC/GGG motif identified so far can be increased to 38.2% (13/34).
In all 17 analysed Slovak AKU patients, both disease causing mutations were found (table 2). The identification of nine different mutations in this sample was not expected because the founder effect had been considered to be the main reason responsible for an increased incidence of AKU in Slovakia. The most frequent mutations, G161R and G152fs (previously identified in two Slovak families by Gehrig et al 6), were present on 50% of 32 screened AKU chromosomes (table 1). So far, these mutations have not been identified in any other screened population. This indicates that they might be specific for Slovakia. The high proportion of these two mutations can be explained by founder effect and subsequent genetic isolation. In addition, however, there must have been at least four other founders contributing to the gene pool of the Slovak AKU population (table 1). Three further mutations were each found on only one AKU chromosome, thus indicating that this mechanism is not the only one responsible for the high incidence of this disease in Slovakia (1:19 000).
Possible common origins of chromosomes carrying the same AKU mutations can be further traced by the analysis of DNA polymorphisms in theHGO gene and construction of haplotypes. This work is now in progress.
Acknowledgments
Links in electronic databases involve: primary structure of HGO from man (HGO, AF00573,15), mouse (MHGO,U58988,16), and Aspergillus nidulans (HMGA, U30797 17), and human HGO transcript (AF045167 5 15).