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Modern Genetic Analysis Chapter 10. Recombinant DNA Technology Using Cloned DNA
Genetics In Process 10-1: A century of genetic research on alkaptonuria

Alkaptonuria is a human disease with several symptoms, of which the most conspicuous is that the urine turns black when exposed to air. In 1898, an English doctor named Archibald Garrod showed that the substance responsible is homogentisic acid, which is excreted in abnormally large amounts into the urine of alkaptonuria patients. In 1902, early in the post-Mendelian era, Garrod suggested, on the basis of pedigree patterns, that alkaptonuria is inherited as a Mendelian recessive. Soon after, in 1908, he proposed that the disorder was caused by the lack of an enzyme that normally splits the aromatic ring of homogentisic acid to convert it into maleylacetoacetic acid. Because of this enzyme deficiency, he reasoned, homogentisic acid accumulates. Thus alkaptonuria is among the earliest proposed cases of an "inborn error of metabolism," an enzyme deficiency caused by a defective gene. There was a 50-year delay before it was shown by others that, in the liver of patients with alkaptonuria, activity for the enzyme that normally splits homogentisic acid, an enzyme called homogentisate 1,2-dioxygenase (HGO), is indeed totally absent. It seemed likely that the enzyme HGO was normally encoded by the alkaptonuria gene. In 1992, the alkaptonuria gene was mapped genetically to band 2 of the long arm of chromosome 3 (band 3q2).

In 1995, Jose Fernandez-Canon and colleagues characterized a gene coding for HGO activity in the fungus Aspergillus nidulans, and, in 1996, they used the deduced amino acid sequence of this gene to do a computer search through a large number of sequenced fragments of a human cDNA library. They identified a positive clone that contained a human gene of 445 amino acids, which showed 52% similarity to the Aspergillus gene. When the human gene was expressed in an E. coli expression vector, it showed HGO activity. Furthermore, when the gene was used as a probe in a Northern analysis of liver RNA, a single RNA of the expected size was hybridized.

When the cloned gene was used as a probe on chromosomes in which the DNA had been partly denatured (in situ hybridizationsee Chapter 12), the probe bound to band 3q2, showing that it was indeed the gene for alkaptonuria.

The cDNA clone was used to recover the full-length gene from a genomic library. The gene was found to have 14 exons and spanned a total of 60 kb. A family of seven in which three children suffered from alkaptonuria was tested for mutations. PCR analysis was used to amplify all the exons individually. The amplified products were sequenced. One parent was heterozygous for a proline serine substitution at position 230 in exon 10 (mutation P230S). The other parent was heterozygous for a valine glycine substitution at position 300 in exon 12 (mutation V300G). All three children with alkaptonuria were of the constitution P230S/V300G, as expected if these were the mutant sites inactivating the HGO enzyme.

(Genetics in Process based on an idea by Charles Scriver.)

© 1999 by W. H. Freeman and Company. All rights reserved.