Mutation and Polymorphism Analysis of the Human Homogentisate 1,2-Dioxygenase Gene in Alkaptonuria Patients

Abstract

Alkaptonuria (AKU), a rare hereditary disorder of phenylalanine and tyrosine catabolism, was the first disease to be interpreted as an inborn error of metabolism. AKU patients are deficient for homogentisate 1,2 dioxygenase (HGO); this deficiency causes homogentisic aciduria, ochronosis, and arthritis. We cloned the human HGO gene and characterized two loss-of-function mutations, P230S and V300G, in the HGO gene in AKU patients. Here we report haplotype and mutational analysis of the HGO gene in 29 novel AKU chromosomes. We identified 12 novel mutations: 8 (E42A, W97G, D153G, S189I, I216T, R225H, F227S, and M368V) missense mutations that result in amino acid substitutions at positions conserved in HGO in different species, 1 (F10fs) frameshift mutation, 2 intronic mutations (IVS9-56G-->A, IVS9-17G-->A), and 1 splice-site mutation (IVS5+1G-->T). We also report characterization of five polymorphic sites in HGO and describe the haplotypic associations of alleles at these sites in normal and AKU chromosomes. One of these sites, HGO-3, is a variable dinucleotide repeat; IVS2+35T/A, IVS5+25T/C, and IVS6+46C/A are intronic sites at which single nucleotide substitutions (dimorphisms) have been detected; and c407T/A is a relatively frequent nucleotide substitution in the coding sequence, exon 4, resulting in an amino acid change (H80Q). These data provide insight into the origin and evolution of the various AKU alleles.

Allelic heterogeneity of alkaptonuria in Central Europe

“…The splice site mutation c.509 + 1G > A affects the same nucleotide as a mutation described in a Dutch patient. 7 However, whereas a G to T transversion was reported there, we have found a G to A transition at the same position. Both base exchanges, however, replace the canonical first G of the consensus donor splice site and are therefore likely to cause skipping of the preceding exon 5.…”

“…In total 10 different mutations were found (Table 1), five of which had been reported in earlier studies. [5][6][7][8] The Gly161Arg (c.648G > A) mutation which we had previously described in a single family 6 turned out to be the most prevalent AKU mutation in both Slovak and Czech patients. It has been observed on 15 of the 38 unrelated AKU chromosomes studied, thus accounting for 39.5% of all disease alleles in the 19 index patients.…”

“…Intragenic DNA markers D3S4556 and D3S4497 were typed according to existing procedures. 7,16 Murine HGO cDNA 13 was cloned into the expression vector pQE-30™ (Quiagen, Hilden, Germany) and transfected into the E. coli strain M15. Site-directed mutagenesis with the QuickChange™ kit (Stratagene, Heidelberg, Germany) was used to introduce human AKU mutations.…”

“…Interestingly, two of the mutations observed in Slovak patients, Pro230Ser and Val300Gly, were reported previously in Spanish, Turkish, French and German families. 7 The spectrum of HGO alleles in the five AKU patients from the Czech Republic overlaps the Slovak mutations with one exception. The splice site mutation c.509 + 1G > A affects the same nucleotide as a mutation described in a Dutch patient.…”

“…5 Inactivating missense and frame-shift mutations established HGO defects as the molecular cause of alkaptonuria in man. [5][6][7][8][9] Independently, the murine HGO cDNA was cloned using amino acid sequence information of the purified enzyme. 10,11 The mutation of the aku mouse, induced by chemical mutagenesis, inactivates a splice donor site.…”