HPRT activity

In many cells, the capacity for de novo synthesis to supply purines and pyrimidines is insufficient, and the salvage pathway is essential for adequate nucleotide synthesis. In patients with Lesch-Nyhan disease, an enzyme for purine salvage (hypoxanthine guanine phosphoribosyl pyrophosphate transferase, HPRT) is absent. People with this genetic deficiency have CNS deterioration, mental retardation, and spastic cerebral palsy associated with compulsive self-mutilation, Cells in the basal ganglia of the brain (fine motor control) normally have very high HPRT activity. These patients also all have hyperuricemia because purines cannot be salvaged. 

The mechanism by which deficiency of HPRT causes central nervous system disorders remains unknown. Lesch-Nyhan patients do not show anatomical abnormalities in the brain. In normal subjects, HPRT activity is high in the brain and, in particular, in the basal ganglia where de novo purine biosynthesis is low. This suggests the importance of the purine salvage pathway in this tissue. However, the relationship between HPRT deficiency and... 

When selecting for loss or gain of HPRT function, the cells should be maintained in the positive or negative selection prior to the selection switch that will assay the altered HPRT activity. The transiently expressed Cre-mediated excision is often mosaic. Therefore, a PCR screen should be carefully designed to detect such situations. Additionally, Southern blot analyses should be performed as a final check on candidate clones identified by PCR. 

In the biochemical analysis of patients with Lesch-Nyhan disease the activity of HPRT found in cell lysates does not always correlate with HPRT activity in the intact cell (1-3). Neither analysis consistently predicts the degree of neurologic impairment in the patient (4,5). 

Evaluation of a patient (D.D.), with many of the neurologic complications of the Lesch-Nyhan syndrome has revealed the most extreme discrepancy we have yet encountered between lysed cell and intact cell HPRT activity. This manuscript will describe the clinical features of the patient and the initial biochemical evaluation of HPRT activity in various cell types. 

Prenatal monitoring of a maternal aunt of D.D. was recently accomplished. The fetus was male and his amniotic cells were normal by radioautography with H-hypoxanthine. Amniotic cell lysates also had normal HPRT activity with 583 nm/mg/hr as compared to the normal range of 528-583 nmole/mg/hr. The normal prenatal diagnosis was confirmed by demonstrating a normal HPRT activity of 59 nmole/ mg/hr in an erythrocyte lysate of cord blood obtained at delivery. 

The extreme disparity between undetectable HPRT activity in cell lysates and mild deficiency in intact cells presents several important problems. On a clinical basis, screening for affected patients by radioautography or other assays involving intact cells can yield a falsely normal result. Furthermore, heterozygote detection by conventional radioautography will not be valid in this... 

Future work will be directed at determining the cause of the discrepancy between the lysate and intact cell assays. The biochemical findings will be applied toward devising an accurate method of heterozygote screening and hopefully toward means of enhancing or stabilizing the HPRT activity in the affected patient. 

The method described here utilizes a radiochemical assay with subsequent thin layer chromatography and autoradiography to measure the HPRT activity in individual hair roots collected from the possible heterozygote. This method is fast and sensitive, and requires no specialized equipment. 

Assay of Resistant Colonies for Levels of HPRT Activity... 

A portion of the cells dispersed from these same resistant colonies was plated into nonselective culture medium and allowed to grow for approximately 10 generations. Cells were then trypsinizcd and plated at cloning densities into three different types of media (1) HAT medium, i.e., Ham s FIO that had been prepared without HX and thymidine but was now supplemented with 30 mM HX (H), 0.1 aminopterin (A), and 30 nM thymidine (T) and 10% FCS (2) nonselective culture medium, i.e., FIO lacking HX but supplemented with 10% FCS and (3) the AG or TG selective medium described in Section 2.1. Growth in HAT medium requires a cell to possess HPRT activity (or, alternatively, to be resistant to aminopterin). Growth in AG or TG selective medium requires a cell to lack HPRT activity or to have altered uptake of these purines. After 3 weeks, the dishes were stained and the number of clones in each set determined. 

Table 1 presents the results of studies characterizing a representative sample of AG- or TG-resistant mutant colonies using the methods outlined in Section 2.4. The results show that the majority of the colonies tested had greatly reduced enzyme levels and that only the positive control cells were able to incorporate HX. One colony exhibited a low level of HPRT activity (6.5% of HPRT cells), yet cells from this colony, after growing for more than 10... 

TABLE 1. HPRT Activity of Randomly Isolated AG- or TG-Resistant Clones... 

Code Cell strain Mutagenic agent Relative HPRT activity % of HPRT+ NF strain) Ability to incorporate [ H]-HX ... 

HPRT activity in iii Ji moles product/mg protein/hour. 

---