A genetic factor for age-related cataract: identification and characterization of a novel galactokinase variant, "Osaka," in Asians

Abstract

Galactokinase (GALK) deficiency is an autosomal recessive disorder characterized by hypergalactosemia and cataract formation. Through mass screening of newborn infants, we identified a novel and prevalent GALK variant (designated here as the "Osaka" variant) associated with an A198V mutation in three infants with mild GALK deficiency. GALK activity and the amount of immunoreactive protein in the mutant were both 20% of normal construct in expression analysis. The K(m) values for galactose and ATP-Mg(2+) in erythrocytes with homozygous A198V were similar to those of the healthy adult control subjects. A population study for A198V revealed prevalences of 4.1% in Japanese and 2.8% in Koreans, lower incidence in Taiwanese and Chinese, no incidence in blacks and whites from the United States, and a significantly high frequency (7.8%; P < .023) in Japanese individuals with bilateral cataract. This variant probably originated in Japanese and Korean ancestors and is one of the genetic factors that causes cataract in elderly individuals.

Figure 1

Pedigrees of families 1–3. Genotype and GALK activity (in nmol/min/g hemoglobin) measured in erythrocytes of male (squares) and female (circles) members of the families. Data in parentheses represent the percentage of GALK activity relative to that in healthy adult control subjects (25.2–40.0 nmol/min/g hemoglobin). The age at which GALK activity was measured is indicated for probands.

Figure 2

Oral galactose–tolerance test in three patients and six healthy control subjects. The oral galactose–tolerance test was performed using a dose of 1 g of galactose/kg of body weight (maximum 50 g). Patient 1, age 1 year; patient 2, age 1 year; patient 3, age 9 mo. Shaded area, mean ± SD data of the healthy control subjects, aged 30–43 years, with normal GALK, GALT, and UDP-galactose 4′-epimerase in erythrocytes. An asterisk (*) denotes a P value <.05, relative to the control subjects.

Figure 3

Identification of missense mutation in patients with the Osaka variant. Each exon and its flanking intronic region was amplified with a pair of human GALK-specific oligonucleotide primers (one primer was biotinylated) using PCR. The amplified products were purified to single-strand DNA, using magnetic beads coated with streptavidin M280 (Dynal), and were sequenced with dye terminator methods using an ABI autosequencer (310 Genetic Analyzer). Primers used for the PCR amplification in exon 4 of the human GALK gene are as follows: biotin-sense primer of 5′-GAATCTCCCTGGAGTGTCATT-3′ and antisense primer of 5′-CAGGCAGTGGGCACACTCCA-3′. Sequencing primer is sense primer of 5′-TCATTGAAGCCACTGCTGCT-3′. The C and T bands appear at the same position in the patients' sequences, resulting in a C→T transition and replacement of alanine by valine at codon 198.

Figure 4

Analysis of GALK mRNA, immunoreactive protein, and GALK activity in COS cells transfected with normal or A198V human GALK cDNAs constructs. A, GALK mRNA levels in cell extracts were determined by dot-blot hybridization for serially diluted samples containing 1, 2, 4, or 8 μg of total RNA with a GALK cDNA probe labeled with [α-³²P] dCTP. The levels of GALK activity and immunoreactive protein were corrected with transfection efficiency by these levels of GALK mRNA. B, Immunoreactive GALK was identified by Western blotting, using a rabbit anti-human GALK antibody, goat anti-rabbit IgG-peroxidase, and ECL system (Amersham Pharmacia Biotech). The density of each band was quantified by scanning with a Bio-Rad Imaging Densitometer (GS-700). C, GALK activity was determined twice to ensure reproducibility using ¹⁴C-galactose and a diethylaminoethyl cellulose column-DE52, as described elsewhere (Shin Buehring et al. 1977). GALK activities in normal, A198V, or mock constructs were expressed as nmol phosphorylated galactose/min/mg protein. The level of GALK activity in the A198V mutant construct was expressed as a percentage between those of normal constructs (100%) and endogenous background (0%).

Figure 5

Allele-specific amplification for A198V mutation. Primers A198-AS for normal and 198V-AS for mutant type had the mismatch at the 3′ end and a deliberate G/A mismatch at three bases from the 3′ ends due to improvement of specificity between normal and A198V mutation. The following primer sets were used for PCR: antisense primer of A198-AS: 5′-CCTGCAGTCAATGAGCAACG-3′ for normal, or of 198V-AS: 5′-CCTGCAGTCAATGAGCAACA-3′ for A198V mutation, and sense primer of 198-S: 5′-GAATCTCCCTGGAGTGTCATT-3′ for normal and A198V mutation. Lane 1, PCR product (213 bp) shows only A198V amplification, indicating that the subject from whom this sample was taken is homozygous for A198V mutation. Lanes 2 and 3, PCR products show both normal and A198V mutant amplifications. The subjects of lanes 2 and 3 are heterozygous carriers of A198V mutation. Lanes 4 to 8, PCR products show only normal amplification. These subjects had the normal genotype. MW = molecular weight marker (100-bp ladder).