Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells.

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.     

Heritable alterations at the adenine phosphoribosyltransferase (APRT) locus in human lymphoblastoid cell lines.

Human lymphoblastoid cell lines derived from WI-L2 exhibit unexpected frequencies of diaminopurine (DAP) resistant mutants. The background mutant fractions of 10(-7) to 10(-8) in untreated cultures are much lower than the frequencies expected for loss of a heterozygous autosomal locus (10(-5) to 10(-6), yet much higher than expected for a homozygous locus (10(-10) to 10(-12). We used aminopterin, adenine and thymidine (AAT) to select DAP-sensitive (DAPS) revertants from one resistant line. The background frequency of DAPR in these revertant cell lines ranged from 3.5 to 6.5 x 10(-4), approximately the square root of 10(-7). Thus these data suggest that both alleles of aprt are inactivated at similarly high frequencies. They also indicate that the DAPS revertants were heterozygotes (aprt +/-) or hemizygotes (aprt +/0) and that WI-L2 was homozygous (aprt+/+). Mutational dose-response studies with X-rays, ethyl methanesulfonate (EMS), and ICR-191 were conducted in 4 of these revertant cell lines. EMS and ICR-191, which induce mainly point mutations, did not induce an increase in mutant fraction. A dose of 200 cGy X-rays, however, induced a frequency of 10(-3). Treatment of DAPR cells with 5-azacytidine induced a significant increase in reversion to DAPS. Southern blot analysis of the aprt gene after digestion with MspI or HpaII also suggests that differential methylation changes may play a major role in the generation of DAP sensitivity and resistance.     

Intervention of somatic mutational events in vivo by a germline defect at the adenine phosphoribosyltransferase locus

Both germline and somatic mutations are known to affect phenotypes of human cells in vivo. In previous studies, we cloned mutant peripheral blood T cells from germline heterozygous humans for adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) deficiency and found that approximately 1.3 × 10^–4 peripheral T cells had undergone in vivo somatic mutations. Loss of heterozygosity (LOH) was the major cause of the mutations at the APRT locus since approximately 80% of the mutant T cell clones exhibited loss of normal alleles. In the present study, we identified three heterozygous individuals for APRT deficiency (representing two separate families), in whom none of the somatic mutant cells exhibited LOH at the APRT locus. The germline mutant APRT alleles of these heterozygotes from two unrelated families had the same gross DNA abnormalities detectable by Southern blotting. None of the germline mutant APRT alleles so far reported had such gross DNA abnormalities. The data suggest that the germline mutation unique to these heterozygous individuals is associated with the abrogation of LOH in somatic cells. The absence of LOH at a different locus has already been reported in vitro in an established cell line but the present study describes the first such event in vivo in human individuals. Received: 10 May 1996     

Rapid method for the diagnosis of partial adenine phosphoribosyltransferase deficiencies causing 2,8-dihydroxyadenine urolithiasis

Summary More than half of the Japanese patients with 2,8-dihydroxyadenine urolithiasis only partially lack adenine phosphoribosyltransferase (APRT), while all the Caucasian patients with the same disease completely lack the enzyme. APRT activities in healthy heterozygotes for the complete APRT deficiencies were at the same levels as the Japanese patients, and simple enzyme assay does not distinguish between these two conditions. We have previously shown, using viable T-cells, that the enzyme was non-functional in the cells from the Japanese patients although they contain considerable APRT activities in the cell extracts. In the present investigations, we devised a rapid method using erythrocytes for the diagnosis of partial APRT deficiencies accompanied by severe impairment in adenine metabolism causing 2,8-dihydroxyadenine lithiasis. Thus, erythrocytes from three different families with 2,8-dihydroxyadenine urolithiasis associated with partial APRT deficiencies incorporated only minimal amounts of radioactive adenine, while normal erythrocytes incorporated significant amounts. These data indicate that severe impairment in adenine metabolism is shown not only in viable T-cells but also in viable erythrocytes. The present procedures provide a rapid method suitable for routine clinical use for the diagnosis of partial APRT deficiencies causing 2,8-dihydroxyadenine lithiasis.     

Identification of two novel mutations in adenine phosphoribosyltransferase gene in patients with 2,8-dihydroxyadenine urolithiasis

Five mutations in the adenine phosphoribosyltransferase (APRT) gene have been described in Japanese patients with APRT deficiency. We investigated the APRT gene from three patients with APRT deficiency and two novel mutations, G133D and V84M, were determined.     

A mutant allele common to the type I adenine phosphoribosyltransferase deficiency in Japanese subjects

Adenine phosphoribosyltransferase (APRT) deficiency is a genetic disorder which causes 2,8-dihydroxy-adenine urolithiasis. The estimated incidence of heterozygosity in Caucasian and Japanese populations is 1%. Mutant alleles responsible for the disease have been classified as APRT*Q0 (type I) and APRT* (type II). In our previous study, we demonstrated in APRT*J a single common base change which accounts for 70% of the Japanese mutants. The present report describes the analysis of an APRT*Q0 mutation in Japanese subjects. Two nucleotide substitutions common to all seven affected alleles from four unrelated subjects (three homozygotes and a heterozygote) were identified: G----A at nucleotide position 1453 and C----T at 1456. The G----A altered the amino acid Trp98 to a stop codon. The C----T did not alter Ala99. These point mutations were demonstrated by sequence analysis of polymerase chain reaction (PCR)-amplified genomic DNA and cDNA. The G----A change at 1453 results in the elimination of a PflMI site in the APRT gene. PflMI digests, which were used to confirm the G----A transition, can be useful in screening for this specific mutation.     

Adenovirus-induced mutations at the hypoxanthine phosphoribosyltransferase locus of Chinese hamster cells.

Hpt-13 is a Chinese hamster cell line deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and sensitive to a medium containing 10(-4) M hypoxanthine, 5.5 X 10(-6) M aminopterin, and 10(-4) M thymidine. In this cell line there is a high incidence of cells resistant to this selective medium after an incubation with either ethyl methane sulfonate or adenovirus type 2 complete virions or their incomplete particles. The rate of reversion in the presence of these agents was 34-fold higher with ethyl methane sulfonate and 2.5- to 5.6-fold higher with adenovirus particles than the spontaneous rate of reversion. The revertant phenotypes were stable for many generations without selective pressure. All of the revertants tested recovered the hypoxanthine phosphoribosyltransferase activity. Most of them, however, carried an enzyme of lower activity and faster electrophoretic mobility than that of the wild type. The preferential reversion to this type of enzyme was observed among spontaneous revertants as well as among those induced by mutagenesis with ethyl methane sulfonate or exposure to viral particles. Our results suggest that adenovirus particles and ethyl methane sulfonate induce mutations at the hpt locus of Hpt-13 cells through similar mechanisms.     

Mutagenicity testing in mammalian cells: I. Derivation of a chinese hamster ovary cell line heterozygous for the adenine phosphoribosyltransferase and thymidine kinase loci

As a first step in the development of a multiple-marker, mammalian cell mutagenesis assay system, we have isolated a Chinese hamster ovary (CHO) cell line that is heterozygous for both the adenine phosphoribosyltransferase (aprt) and thymidine kinase (tk) loci. Presumptive aprt^+/? heterozygotes with intermediate levels of APRT activity were selected from unmutagenized CHO cell populations on the basis of resistance to low concentrations of the adenine analog, 8-azaadenine. A functional aprt^+/? heterozygote with ～50% wild-type APRT activity was subsequently used to derive sublines that were also heterozygous for the tk locus. Biochemical and genetic characterization of one such subline, CHO-AT3-2, indicated that it was indeed heterozygous at both the aprt and tk loci. CHO-AT3-2 cells permitted single-step selection of mutants resistant to 8-azaadenine or 5-fluorodeoxyuridine, allowing quantitation and direct comparison of mutation induction at the autosomal aprt or tk loci, as well as in the gene involved in ouabain resistance or at the X-linked, hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus. Significant dose-dependent increases in mutation frequency were observed for all 4 genetic markers after treatment of CHO-AT3-2 cells with ethyl methanesulfonate.     

The origin of the most common mutation of adenine phosphoribosyltransferase among Japanese goes back to a prehistoric era

The incidence of adenine phosphoribosyltransferase (APRT) deficiency is higher among Japanese nationals than among other ethnic groups, and the most common mutation (APRT*J, ATG to ACG mutation at codon 136) accounts for 68% of the disease-causing genes among Japanese. To investigate the origin of these mutations, we studied the geographical distribution of the mutant genes in Japan. The APRT*J mutation is distributed nearly uniformly in the four main islands of Japan and Okinawa, suggesting a very early origin. The products of PCR amplification between positions 2344 and 2750 of the genomic APRT sequence were examined by SSCP analysis in random blood samples from Japanese, Korean, and Taiwanese nationals. Among 955 random Japanese blood samples, 7 (0.73%) were heterozygous for the APRT*J mutation, giving a calculated heterozygote frequency of 1.1% among Japanese for the entire APRT deficiency. None of 231 Taiwanese samples contained heterozygotes for the APRT*J mutation, while 2 (0.53%) of 356 Korean samples were heterozygous. In addition to the APRT*J sequence, a total of five variant sequences was found. Sequencing one variant revealed a base substitution in intron 4, suggesting therefore that they are harmless mutations. Since the APRT*J mutation is present in Koreans and Okinawans who share ancestors only before the Yayoi era (third century bc to third century ad), the origin of the APRT*J mutation predates 300 bc. Received: 14 May 1996     

Identification of a single missense mutation in the adenine phosphoribosyltransferase (APRT) gene from five Icelandic patients and a British patient.

We have completely sequenced the adenine phosphoribosyltransferase (APRT) gene from each of six patients--five (I-V) from Iceland and one (VI) from Britain. Cases I and II shared a common ancestor six and seven generations ago, and cases I and V shared a common ancestor seven generations ago, but cases III and IV were unrelated to the above or to each other, over seven generations. Genomic DNA was amplified by PCR, subcloned into M13mp18, and sequenced. Genomic and PCR-amplified DNAs were also analyzed by restriction-enzyme digestion and Southern blotting. The same missense mutation was identified in all six patients. This mutation leads to the replacement of asp (GAC) by val (GTC), at amino acid position 65. The gene sequences from all patients were otherwise identical to our wild-type sequence. The homozygous nature of the mutation was confirmed by sequencing the PCR product directly. All six patients were homozygous for the 1.25-kb TaqI RFLP. The Icelandic patients were also homozygous for the 8-kb SphI RFLP, but the British patient was heterozygous at this site. These studies suggest that a founder effect is likely to be responsible for APRT deficiency in the Icelandic population. The finding of the same mutation in a patient from Britain suggests that this mutation may have originated in mainland Europe.     

Identification of a compound heterozygote for adenine phosphoribosyltransferase deficiency (APRT*J/APRT*Q0) leading to 2,8-dihydroxyadenine urolithiasis

Summary Homozygous deficiency of a purine salvage enzyme, adenine phosphoribosyltransferase (APRT), causes urolithiasis and renal failure. There are two known types of homozygous APRT deficiencies; type I patients completely lack APRT activity while type II patients only partially lack such activity. All type II patients possess at lest one APRT^*J allele with a substitution from ATG (Met) to ACG (Thr) at codon 136. Type I patients are considered to possess two alleles (APRT^*Q0) both of which code for complete deficiencies. Thus, some patients with type II APRT deficiencies may have a genotype of APRT^*J/APRT^*Q0. As no individuals with such a genotype have previously been identified, we performed extensive analysis on four members of a family by (1) the T-cell method for the identification of a homozygote, (2) the B-cell method for the identification of heterozygotes, and (3) oligonucleotide hybridization after in vitro amplification of a part of genomic APRT sequence for the identification of APRT^*J and nonAPRT^*J alleles. We report here the first evidence that 2,8-dihydroxyadenine urolithiasis developed in a boy aged 2 years with a genotype of APRT^*J/APRT^*Q0.     

Accelerated development of uroporphyria in mice heterozygous for a deletion at the uroporphyrinogen decarboxylase locus

Three weeks after a single dose of iron-dextran and Aroclor 1254, mice maintained continuously on delta-aminolevulinic acid supplemented drinking water showed significantly elevated levels of hepatic uroporphyrin and depressed (25% of normal) uroporphyrinogen decarboxylase (URO-D) activity. Depressed URO-D activity was paralleled by the ability of heat denatured cytosol to inhibit rhURO-D activity. Mice heterozygous for a targeted disruption at the URO-D locus (URO-D+/-) exhibited half the URO-D activity of homozygous controls prior to treatment. After treatment, these animals showed URO-D activity and rhURO-D inhibitory activity comparable to similarly treated wild type (URO-D +/+) mice but with significantly greater uroporphyrin accumulation. With only 10 days of treatment, URO-D +/- but not URO-D +/+ mice showed changes similar in magnitude to those seen after 21 days. Prior to treatment, URO-D genotype did not influence overall hepatic P450 concentration in either sex and there was no significant difference between sexes. The treatment regimen significantly elevated P450 in animals of either URO-D genotype and in both sexes, although the induction response at the 10-day point was attenuated in URO-D +/- mice. From differences in the CO absorbance maximum, and by P450 activity analysis, this attenuated induction response resulted from an attenuation of the CYP2B not the CYP1A induction.     

Selection at the ABO locus in the Japanese population

Hiraizumi et al. (1973b) concluded that maternal-fetal ABO incompatibility effect upon the frequency of prenatal deaths was totally absent in two modern Japanese cities, Ohdate and Akita. However, they found a significant heterogeneity in the frequency of prenatal deaths among 16 mating types and suggested that the viability of A-bearing fetuses was higher than that of others, hence the frequency of prenatal deaths decreased with the increasing probability of producing A-bearing fetuses. Further analyses were performed in the present study and the above suggestion was confirmed. Then, the average numbers of prenatal deaths and pregnancies per couple were analyzed separately for their relationships with the probability of producing an A-bearing fetus. The results were found to be consistent with the model that the average numbers of prenatal losses per couple are approximately the same between two groups of matings, one producing and the other not producing A-bearing fetuses, but such losses occur more frequently for the A-bearing fetuses than others at the very early stage of pregnancy such that wives may not recognize such losses. Although the stages of losses are different between A-bearing and other fetuses, the net losses are nearly the same between them, so that such losses will not be reflected in the segregation frequencies among children, as shown by Hiraizumi et al. (1973a).