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INTRODUCTION

The gene coding for HGPRT, or Hypoxanthine guanine phosphoribosyltransferase is found on the long arm of the X chromosome, q26-q27.2. (See Figure 1) This 57kb nucleotide long gene is composed of 9 exons (in blue) and 8 introns (in white) and is transcribed into a 1.6kb mRNA containing a 654 nucleotide protein encoding region. (Sculley) Three pseudogenes have been located on chromosomes 3, 5 and 11, have been identified (Stout and Caskey, 1984).

The arrows indicate where point mutations have been known to lead to the Lesch-Nyhan Syndrome (will be discussed later).

As a housekeeping gene, HGPRT is consitutively expressed in all tissues but notably in the mammalian brain. In fact, in a study of mouse brains, a high level of HGPRT activity was observed in the hippocampus which is involved in learning and memory in humans. In the mouse brains, low HGPRT activity was detected at birth. Seven days after birth there was a rapid increase until a few days after when the activity gradually increased to mature levels. Such observations were seen especially in the hippocampus. Through this prenatal analysis, scientists were able conclude that HGPRT probably has an effect on the development of the hippocampus. (Nakagawa) The specific role of the HGPRT enzyme is in the purine salvage pathway.

THE ROLE OF HGPRT IN PURINE METABOLISM

Cells are able to make purines by the use of two pathways- the de novo pathway and the salvage pathway. In the de novo pathway, amino acids, CO2, NH3, and ribose-5-phosphate are used to make purines whereas in the salvage pathway, the cells recycle free purine bases. The latter pathway is a less expensive reaction than the de novo pathway. Three reactions in the salvage pathway are catalyzed by two salvage enzymes; (1) the conversion of adenine plus PRPP (5-phosphoribosyl-1-pyrophosphate) to adenylate plus PPi by the enzyme adenine phosphoribosyl transferase and (2+3) the conversions of hypoxanthine and guanine plus PRPP to IMP and GMP respectively, and PPi by the action of HGPRT. The last two reactions are partially regulated by feedback inhibition in the presence of IMP and GMP. Purine metabolism is quite important in the brain hence we can theorize that the function of HGPRT is very important in the body. In fact as it shall be discussed later, a deficiency in HGPRT may have devastating results. See Figure 2 for biochemical summary of purine synthesis.

(Pathways of purine synthesis) Figure 2.

DETERMINING THE LOCATION OF THE HGPRT GENE

The genetic map of the HGPRT gene as shown above, was determined through several techniques including linkage analysis, restriction endonuclease techniques and by the use of somatic cell hybrids. (OMIM) The first X-linkage analysis of HGPRT was done 1965 by Hoefnagel et al. In linkage analysis, recombination frequencies are measured in families. From the numbers, it was possible to determine the position of the gene on the X chromosome relative to other genes and/or the centromere. Restriction endonuclease techniques combined with somatic cell hybridization is also a way to determine the location of a gene. (D. Weil et al) In somatic cell hybridization, mouse cells and human cells are fused together. Eventually the nuclei also fuse together and the cells become a hybrid. As the cell divides, it's has been found that human chromosomes are lost. It is easy to differentiate human and mouse chromosomes under a microscope hence it is possible to determine which chromosome is lost. Following the analysis of the presence and absence of specific genetic markers relative to the presence and absence of certain human chromosomes, scientists are able to figure out where a gene is located.

HGPRT AS A RECESSIVE SEX LINKED GENE

HGPRT is passed on as a recessive sex linked trait. A nonfunctional version of this gene would affect males more than females as the effects of the gene would be masked by the normal X chromosome in the female. In females, one of the X chromosomes is inactivated and forms a Barr body. This is called random X chromosome inactivation. Thus, a female will be a mosaic of cells containing one or the other inactive X chromosome. Hence in a heterozygous HGPRT female, some of cells will be HGPRT deficient and some will not however overall the female will be healthy. Studies have shown that the levels of HGPRT in a heterozygous female is lower than that of a homozygous HGPRT+ female. On the other hand, all the cells in a male will be either normal HGPRT or HGPRT deficient and hence the male will feel a full effect of a defective gene.

X linked recessive inheritance can be observed by analyzing the offspring of a "normal" heterozygous female and a "normal" homozygous male. We would notice that only (or mainly) males express the recessive phenotype. See the following pedigree for an example. It is to be noted that the heterozygous female does not show the recessive phenotype unlike her homozygous defective gene brother. In the case of HGPRT deficiency, the affected males do not reproduce hence it is very rare to see a homozygous female for this defective gene.

THE EFFECTS OF A DEFECTIVE HGPRT GENE IN HUMANS: (Lesch-Nyhan Syndrome, Kelly-Seegmiller syndrome, and Gout)

The HGPRT gene may become nonfunctional by different types of mutations. Up to now, more than 50 mutations have been described in this gene. Some of the mutations include deletions, frameshift errors, and transitions. These mutations can cause the HGPRT protein to be either completely nonfunctional or only partially nonfunctional. In humans, partial HGPRT deficiency may lead to Kelley-Seegmiller syndrome or gout whereas a complete HGPRT deficiency may lead to Lesch-Nyhan syndrome.

A defective HGPRT protein would cause less IMP and GMP to be created by the salvage pathway. The consequence would be an accumulation of PRPP, an important molecule required for the synthesis of purines, and an increase in the de novo pathway to make the required purines. An over production of the purines would then cause an accumulation of uric acid in the blood when broken down. Uric acid is toxic in high levels and it causes damage to tissues. In fact, some of symptoms of a deficiency in HGPRT is the spontaneous formation of uric acid crystals in urine that is left standing and the deposition of uric acid in joints or kidneys.

Lesch-Nyhan syndrome affects about 1 in every 400,000 children and the effects are quite severe. Mental retardation, severe self mutilation and high levels of uric acid in urine are some of the symptoms.

It is interesting to note that HGPRT- mice show the same metabolic defects as humans however they do not seem to demonstrate the human behavioral abnormalities. (Patel)

By sequencing HGPRT cDNA and genomic DNA, scientists have been able to find several mutations which causes Lesch-Nyhan syndrome. One of these mutations was a single point mutation in exon 3 which resulted in a conversion from a guanine to a thymine. This resulted in abnormal mRNA splicing that gave two types of mRNA. One type of splicing gave rise to a protein with 22 amino acids deleted and another gave rise to a protein lacking 3 amino acids. (Yamada et al)

A rare case of a female with Lesch-Nyhan showed that the female's gene had been inactivated by a novel nonsense mutation. As the mother's cDNA was normal, scientists concluded that the mutation must have been due to a de novo gametic event in the paternal gene. Secondly, they demonstrated that the X-chromosome inactivation in this patient was non random favoring the maternal gene. This is an interesting example because it shows how a X-link recessive trait can be found in a female.

DETECTION OF DEFECTIVE HGPRT GENE

Lesch-Nyhan syndrome carriers can be detected by several methods such as autoradiography which measures the HGPRT activity in fibroblasts, mutations detection by PCR and direct sequencing and linkage analysis. Prenatal diagnosis can be done using amniotic fluid cells and measuring the enzyme's activity. The syndrome may be recognized in a fetus well before 20 weeks. (OMIM)

Another method used in estimating the activity of HGPRT is by spectrophotometry. This assay measures HGPRT activity in erythrocyte and lymphocyte lysates and in brain homogenates. (D. T. Keough)

A CURE FOR LESCH-NYHAN SYNDROME?

At first glance, one might ask: why don't we just provide the HGPRT enzyme to patients suffering from a deficiency? Furthermore, patients don't need very much HGPRT to make a very large difference. However this treatment is not possible as the HGPRT enzyme is large and can not get through the defense system or if take orally will be broken. So far there is no cure for Lesch-Nyhan syndrome however several avenues are being studied including the use of gene therapy and inhibitors of the de novo pathway.

THE USE OF HGPRT IN CELL BIOLOGY EXPERIMENTS

HGPRT has been a valuable gene to work with. One use of this gene is for selection of hybrid cells. For example, a cancer cell that is deficient in HGPRT is fused to a normal cell that is HGPRT+ but TK- . The fusion of these two cells will create a hybrid that has both functional HGPRT and TK. TK stands for thymidine kinase and is required for the cells to grow. The hybrids are grown on HAT medium which contains Hypoxanthine, Aminopterin, and Thymine. Aminopterin blocks the de novo pathway forcing the cells to use the salvage pathway. Thus, cells that are HGPRT- or TK- cannot grow on HAT medium and only the hybrids cells will grow on the medium. See Figure 3.

CONCLUSIONS

The HGPRT gene is an interesting example of how a single mutation can have disastrous effects on someone's health. More studies are needed to help cure patients suffering from the effects of these mutations. The gene is also very useful in helping cell biologists make monoclonal antibodies and other experiments so knowing more about a gene is always beneficial.

URLs:

Purines and Pyrimidines

Lesch-Nyhan

Genetic Diseases

Genetics research

UCSD Biochemical Genetics Local Test Listing

Screening for mutations in HGPRT cDNA by use of PCR

Cloning and sequencing of cDNA for mutation analysis

Gene HPRT1

Bioch 5853: Minireviews part 2: Inborn Errors in Metabolism

Purine Research Society

Nucleotide Metabolism

OMIM ENTRY 308000

Hprt, hypoxanthine guanine phosphoribosyl transferase

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Last updated July 23, 1999