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Sequence analysis of the homogentisate 1,2 dioxygenase gene in a family affected by alkaptonuria
  1. K WALTER,
  2. A GAA,
  1. Institute for Pathology, University of Freiburg, Freiburg, Germany
  2. Institute for Genetics, University of Cologne, Cologne, Germany
  1. Dr Walter, Department of Surgery, University Hospital, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany.
  1. A ROERS
  1. Institute for Pathology, University of Freiburg, Freiburg, Germany
  2. Institute for Genetics, University of Cologne, Cologne, Germany
  1. Dr Walter, Department of Surgery, University Hospital, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany.

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Editor—Alkaptonuria (AKU) is a disorder of the catabolism of aromatic amino acids. A defect of homogentisate 1,2 dioxygenase (HGO) leads to an accumulation of homogentisic acid (HGA) and subsequently to deposition of polymerised HGA, a brown-black pigment, in connective tissue, primarily in cartilage.1 2This phenomenon is known as ochronosis. It results in debilitating arthropathy which typically becomes manifest in the fourth decade of life. Large amounts of HGA are excreted in the urine and cause its black discolouration upon oxidation. In 1891, homogentisic acid was first isolated by Wolkow and Baumann3 from the urine of an AKU patient from a remote area of the Black Forest in south western Germany. In 1902, Garrod, aware of this biochemical finding, observed the autosomal recessive mode of inheritance of AKU and thereby showed for the first time that mendelian laws also apply to human genetics.4 Garrod postulated that AKU results from an enzyme deficiency and introduced the concept of the “inborn error of metabolism”.5

Recently, the human gene encoding HGO was cloned by Fernádez-Cañón et al.6Two different mutations of this gene were identified in two unrelated AKU affected families. These mutations cosegregated with manifest disease and could be shown to abrogate enzymatic activity of HGO protein.6 Homozygosity for these mutations, therefore, was the cause of AKU in the two families. Two additional mutations in the HGO gene were found to cosegregate with AKU in two Slovakian pedigrees.7 One of these mutations caused a frameshift in an upstream exon and was thus likely to result in a loss of HGO activity. For an additional mutation, complete cosegregation with AKU was reported in an extensively studied Canarian family.8Various different mutations of the HGO gene were found in 14 unrelated AKU patients.9

We performed sequence analysis of the HGO gene in an AKU affected family from the Black Forest. AKU with severe ochronosis including involvement of the sclerae was diagnosed at necropsy of a 71 year old farmer (fig 1, No 1). The diagnosis of AKU had not been established during the patient’s lifetime. He died of recurrent myocardial infarction. Subsequently, the patient’s family underwent physical examination. A sister (fig 1, No 2) and a first cousin (fig 1, No 3) were found to be affected by the disease. These patients have been suffering from arthritic symptoms of AKU since the fourth decade of life and show the typical discolouration of the urine and the ochronotic pigmentation of the sclerae.

Figure 1

AKU affected family. Sequence analysis was performed for subjects 1-7. For patients 8 and 9, the family reported AKU related symptoms. They were, therefore, probably affected. Four consanguineous marriages in the last three centuries are indicated by Roman numerals. An asterisk marks the person who comes from the same kindred as the patient of Wolkow and Baumann.3

However, the condition had until then been misdiagnosed as degenerative polyarthritis. A brother (fig 1, No 4) of patient 1 was healthy as were the three children (fig 1, Nos 5, 6, and 7) of patient 2. Anamnestically, a brother (fig 1, No 8) and a first cousin (fig 1, No 9), who died in 1988 and 1995, respectively, were reported to have suffered from debilitating early onset polyarthropathy and the typical ochronotic involvement of the sclerae. They were very probably affected by AKU. No characteristic AKU symptoms were reported for two brothers (fig 1, Nos 10 and 11) of patient 1, who died in 1940 and 1974, respectively.

The pedigree of the family was constructed using the parish registers of the region. Information was available back to the early 17th century. In the last three centuries the pedigree shows four consanguineous marriages of third and fourth cousins (fig 1, I), fifth and seventh cousins (fig 1, II), second and third cousins (fig 1, III), and third cousins (fig 1, IV).

DNA was extracted by standard procedures from liver tissue sections obtained during necropsy of patient 1 and from blood samples of two other affected subjects (Nos 2 and 3) and four healthy members of the family (Nos 4, 5, 6, and 7). The 14 exons of theHGO gene were amplified using primers hybridising in the flanking intronic sequences, as described by Fernández-Cañón et al,6and PCR products were directly sequenced from both sides.

Sequence analysis of the HGO gene showed that all three family members suffering from AKU were homozygous for a point mutation in exon 13, an adenine for guanine substitution at position c1269 (c1269A→G), which causes the replacement of methionine 368 by valine. The three healthy children of patient 2 and the healthy brother of patient 1 were heterozygous carriers of this mutation. Interestingly, the same mutation of the HGOgene was recently described in a French and another German AKU affected family.9 Methionine 368 is conserved betweenAspergillus nidulans, mouse, and man and may be essential for enzymatic activity of HGO protein.10 11

It is of anecdotal interest that our patients stem from the same remote, in former times genetically isolated, area of the Black Forest as the AKU patient from whose urine Wolkow and Baumann3first isolated homogentisic acid in 1891. Therefore, it is likely that their patient was homozygous for the defectiveHGO allele we found in our AKU family.


The authors thank Andrea Klöckner for expert technical assistance and Oskar Hoog for help with the genealogical study.