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Alkaptonuria in Italy: polymorphic haplotype background, mutational profile, and description of four novel mutations in the homogentisate 1,2-dioxygenase gene
  1. * Dipartimento di Fisiopatologia Clinica, Università degli Studi, Firenze, Italy
  2. Divisione di Reumatologia, Azienda Sanitaria Locale, Firenze, Italy
  3. Dipartimento di Pediatria, Università degli Studi, Firenze, Italy
  4. § Departamento de Inmunología, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain
  5. Unidad de Patología Molecular, Fundación Jiménez Díaz, Madrid, Spain
  1. Dr Porfirio, Department of Clinical Physiopathology, Human Genetics Unit, Viale G Pieraccini 6, I-50139 Florence, Italy, n.porfirio{at}

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Editor—Alkaptonuria (AKU, OMIM 203500) is a rare disorder caused by the deficiency of homogentisate 1,2 dioxygenase (HGO, EC HGO catalyses the conversion of homogentisate (HGA) to maleylacetoacetate in the phenylalanine/tyrosine catabolic pathway.2 As a consequence, affected subjects excrete HGA in their urine, which becomes dark upon exposure to air. The medical interest in this condition stems from its association with ochronosis, or the deposition of a brownish pigment in connective tissues including cartilage, where its accumulation can produce a debilitating degenerative joint disease.3

AKU occupies a unique place in the history of human genetics because it was the first disorder to be described as a Mendelian recessive trait.4-6 Recent advances in the understanding of the molecular basis of AKU7-9 have verified that loss of function mutations in the HGO gene are responsible for the disease. A few mutations have been repeatedly detected in patients from different European countries. Since these mutations segregated with specific haplotypes, they should be considered to be old mutations that have spread throughout western Europe with migration. However, allelic heterogeneity is the main feature emerging from the above and other studies.7 9-12Most HGO mutations were found in just one family and did not involve CpG dinucleotides. Rather, a preferential occurrence in the CCC sequence motif and its inverted complement GGG has recently been reported.12 Furthermore, some AKU chromosomes escaped mutation detection within theHGO coding region, suggesting the existence of HGO alleles whose defect might be related to gene expression.

To determine the extent of allelic heterogeneity in Italian patients, we started a systematic search of AKU families through announcements at relevant National Congresses. We present here the results leading to the identification of four novel mutations. Our data should facilitate future mutation screening in Italian AKU patients and carrier identification by DNA typing.

Ten affected subjects from five unrelated Italian AKU pedigrees were included in the study. In three families it turned out that the patients' parents were consanguineous. Three patients were diagnosed at birth as having AKU through analysis of homogentisic aciduria. The remaining cases were diagnosed in adulthood on the basis of clinical and radiological examination. Seventeen normal relatives were also investigated. Both genomic DNA and HGO cDNA were obtained from peripheral blood by standard methods.

RNA was extracted from peripheral blood lymphocytes by the guanidinium thiocyanate-phenol-chloroform method.13 cDNA synthesis was performed using both oligo dT and specific primer mapping in the 3′UTR of the HGO gene. From the complete sequence of a human HGO cDNA (AF045167), primers were designed to obtain overlapping amplicons spanning the entire coding sequence of the HGO gene by nested PCR, as described previously.14 15 Primers HTEL and HTER were used in the first PCR, whereas each of the primer pairs HS1L/HS9, HS4/HS7, HS6/HS3F, HS8/HS12, HS11F/HS10, HS13/HS5 were used in the second PCR. The primer sequences and the expected lengths of the PCR products are shown in table 1.

Table 1

PCR primers for nested PCR analysis of HGO cDNA from blood lymphocytes

SSCP analysis was performed according to Orita et al. 16 PCR products were heat denatured and subjected to electrophoresis on a non-denaturing 6% polyacrylamide gel and silver stained.17 All cases were amplified starting from differently obtained cDNA preparations and run independently at least twice with consistent results.

Genomic DNA was used to amplify the exons included in the cDNA regions where abnormal SSCP patterns were found. Moreover, failure to detect mutations in three families necessitated that the genetic lesions were sought at the genomic level by SSCP screening of each exon amplified with intron primers.9 PCR products were purified by column filtration and sequenced directly with a dye terminator cycle sequencing kit (ABI PRISM Perkin Elmer, Norwalk, USA) using the ABI 377 automated sequencer (Applied Biosystems, Foster City, USA) and its associated analysis software.

Three intragenic STRs, HGO-1 (D3S4496, intron 4), HGO-2 (D3S4497, intron 13), and HGO-3 (D3S4556, intron 4), have been described previously8 9 and were analysed by PCR with modifications to comply with non-isotopic detection. The PCR products were run on denaturing 6-8% polyacrylamide gels and the alleles were visualised by silver staining. The alleles were numbered consecutively and sized by comparison with known samples. Four SNPs, IVS2+35T/A, IVS5+25T/C, IVS6+46C/A, and c407T/A (exon 4), have also been described9 and were studied by SSCP on non-denaturing 8% polyacrylamide gel electrophoresis followed by silver staining. Genotype assignment was made possible by comparison with known samples. Familial segregation provided unequivocal derivation of the haplotypes present on the AKU chromosomes.

Five additional unrelated AKU families were analysed for mutations and polymorphisms in the HGO gene. The IVS9-56G→A and IVS9-17G→A HGO mutations in one Italian patient have already been described.9Therefore, the mutations found in 12 AKU chromosomes of Italian ancestry are presented here (table 2). Since consanguineous marriage occurred in three families, only nine chromosomes may be considered to be independent in origin. In fact, patients from these families were homozygous for the AKU mutations G152fs (c621insG), G270R (c975G→A), and G360R (c1245G→C), respectively. The AKU patients in another family were compound heterozygotes for K248R (c909A→G) and IVS7+5G→A (c636+5G→A). Finally, in family VRN, the AKU patient is most likely a compound heterozygote for G360R and an as yet unknownHGO mutation. Therefore, as many as eight different HGO mutations were found, four of which were novel. We also anticipate that the AKU mutation that remains to be identified will be novel (table 2) because we know that it is different from all previously characterised AKU mutations. We provisionally denoted this mutation as VRN. Three of the four novel mutations (K248R, G270R, and G360R) are missense mutations that affect HGO amino acid residues that are conserved in different species and are likely to be loss of function mutations. K248R is the consequence of an A to G transition at position c909 in exon 10, G270R results from a G to A transition at position c975 in exon 11, and G360R is caused by a G to C transversion at position c1245 in exon 13. This latter mutation was found twice in two unrelated patients. The other novel AKU mutation (IVS7+5G→A) is a G to A transition in the fifth nucleotide position of the donor (5′) splice site sequence of intron 7, which most likely causes aberrant splicing of HGO. None of these four novel AKU mutations were observed, using SSCP screening, in a sample of 100 control chromosomes. Finally, the mutation G152fs originated from a one base insertion at position c621, which determined a frameshift eventually leading to premature arrest of the protein synthesis. This same mutation was described in two Slovak families.10 Although the loss of function nature of all these HGO mutations was not formally proven, SSCP and sequence analysis of the relevant DNA fragment in family members confirmed in all cases that the pattern of inheritance of AKU was compatible with the segregation of theHGO mutations.

Table 2

HGO mutations and haplotypes identified in Italian AKU patients

We managed to perform SSCP analysis of cDNA fragments from two families, one where the G360R mutation was segregating and the other where the patients were compound heterozygotes for the K248R and the IVS7+5G→A mutations. It is interesting to note that this latter supposedly splice site mutation did not affect the SSCP pattern of the cDNA amplicon defined by primer pair HS6/HS3F spanning exons 7 to 9. Furthermore, IVS7+5G→A seemed to have no influence on HGO mRNA stability, as judged by the presence of both wild type and mutant bands in the cDNA amplicon defined by primer pair HS8/HS12 containing the K248R site.

In the light of the recent report of a preferential occurrence ofHGO mutations in the CCC/GGG sequence motifs,12 we analysed the sequence context of the four novel mutations found in the present work. It could not be coincidental that G270R and G360R mutations take place in tri- and penta-G runs, respectively. Moreover, the G152fs mutation previously described in two Slovak families10 occurs in a tetra-G run. Whether the G152fs mutation we found in an Italian family has an independent origin remains to be determined. Haplotyping of the Slovak pedigrees as well as comparison with the Italian one could provide strong evidence that the CCC/GGG motif is a mutational hot spot inHGO.

Table 2 also shows the haplotypes at theHGO intragenic markers IVS2+35T/A, c407T/A, HGO-3, HGO-1, IVS5+25T/C, IVS6+46C/A, and HGO-2 which are associated with each AKU chromosome. Analysis of theHGO haplotypes harbouring the purported causative AKU mutations showed that the three intronic mutations were found in a common haplotypic background composed of the very same alleles at four SNPs as well as at HGO-1. Beltrán-Valero de Bernabéet al 9 referred to this as haplotype A, the most frequent in European populations. Two other mutations, G152fs and G270R, were detected in a gametic association, haplotype D,9 which is derived from haplotype A by variation at the SNP IVS2+35T/A. Closely related to haplotype A is also that segregating with mutation G360R and that harbouring the mutation K248R, haplotype B.9 These haplotypes differ from haplotype A at SNPs c407T/A and IVS6+46C/A, respectively. On the other hand, the mutation VRN was probably within the so-called haplotype E.9 It has been postulated9 that haplotype E has a North African origin, an ethnic component that is known to have contributed to the modern Italian population.18 Whether the VRN mutation has originated in Italy or has been introduced into this country with the different migrations is at present unknown.

Only two of the AKU mutations found in Italians (G152fs and IVS9-56G→A) have been encountered in patients from other European countries. One of them, as indicated before, is the G152fs mutation that was identified in two Slovak AKU pedigrees.10 It would be interesting to determine whether the G152fs mutation has an eastern European origin and appeared in Italy by migration. The IVS9-56G→A AKU mutation was also identified earlier in an AKU chromosome of a French patient.9 Interestingly the IVS9-56G→A mutation in the Italian and the French patients are associated with the same HGO haplotype. In this case, we postulate an Italian origin for this mutation since the French IVS9-56G→A carrier patient has Italian ancestors.

It is important to note that the mutations P230S, V300G, and M368V, which are relatively common mutations,7 12 15 19 were absent in our patients. P230S and V300G are typically associated with haplotype E and are thought to be North African in origin.9 The M368V mutation is widely distributed throughout Europe and is associated with haplotype A.9 It is also interesting that the mutation G360R was found twice within the same haplotype in two families from different ends of the Italian peninsula; patient VRN was from Calabria (southern Italy), whereas the affected sons of a consanguineous marriage were from South Tyrol, a German speaking region on the Austrian border. AdditionalHGO mutational and polymorphism analyses of AKU patients from many more different countries would be necessary to understand the population genetics of AKU and the migration of the different AKU alleles.

In conclusion, we report here an extensive description of the spectrum of AKU mutations in Italy, including the characterisation of their associated intragenic HGO polymorphisms. Four novel mutations were found, which include both missense mutations and subtle intronic changes. The Italian AKU sample confirms the high degree of allelic heterogeneity of the HGOgene and illustrates the complications of mutation screening in AKU patients. These data should facilitate the future identification of these AKU alleles in this and other populations.

Note added in proof

The mutation G270R has recently been described by Mülleret al (Eur J Hum Genet 1999;7:645-51).


We thank Pier Luigi Mattiuz and Umberto Serni for encouragement and suggestions. We are also grateful to Alfredo Brusco and to Claudio Castellan for helping us to identify AKU patients and families. This work was supported in part by a grant from the University of Florence, MURST ex 60% funds, by the Fundación Jose Antonio de Castro, the Spanish Comisión Interministerial de Ciencia y Tecnología (SAF96/0055, SAF97/1789E), and the Comunidad de Madrid (08.6/0015/1997). In addition, this study is based on work supported by the Fundación Conchita Rábago de Jiménez Díaz under a grant awarded to DB-VdeB.