A novel point mutation associated with alkaptonuria

To the Editor: Alkalptonuria (AKU) is a rare autosomal recessive disorder resulting from loss of homogentisate 1,2dioxygenase (HGO; EC 1.13.1 1.5) activity and has existed for more than 3000 years in humans (1, 2). The locus (symbol AKU) was assigned to chromosome 3q2 (2-4). The HGO complementary DNA (cDNA) was cloned by Fernandez-Canon et al. ( 5 , 6). They revealed for the first time that AKU is caused by loss of HGO activity at the molecular level. One point mutation, Pro230Ser in exon 10, has been determined responsible for this disease either because HGO is not correctly expressed or because the encoded polypeptide lacks enzymatic activity. Another point mutation, Va1300Gly in exon 12, may also be responsible for this disease (1). Now, we report a novel point mutation which is associated with a Japanese AKU family. A 40-year-old woman was admitted to our hospital because of ‘lumbago and black urine’. Degenerative arthritis in the spine was observed on a roentgenogram and a high level of urinary homogentisic acid (HGA: 5.5 mmol/l, normal value: 0 mmol/I) was detected by high performance liquid chromatography. The patient was diagnosed as having alkaptonuria. Her parents had been healthy and had no history of having any medications. They had died of cerebrovascular diseases. but their marriage was not consanguineous. The patient was married and had three healthy children. The patient, her three children and five healthy volunteers were selected for this study. The DNA of peripheral white blood cells from the four family members and five healthy volunteers was extracted using an IsoQuick nucleic acid extraction kit (ORCA Research Inc., Bothell, WA, USA). The entire sequence of the HGO gene from exon 1-14 was amplified using a series of primers and a polymerase chain reaction (PCR) amplification kit (Takara, Shiga, Japan). After confirming the sizes and homogeneity of the PCR products by agarose gel electrophoresis, direct sequencing of the entire coding region of the HGO gene was performed using an ABI PRISMTM dye terminator cycle sequencing core kit (Perkin-Elmer, Foster