Gene/Protein Disease Symptom Drug Enzyme Compound
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The Lesch-Nyhan disease is caused by an almost complete lack of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Partial HPRT-deficiency, associated with less severe phenotype, has also been identified. We have characterized mutations occurring in HPRT cDNA isolated from patients with HPRT-deficiency with an emphasis on examining the more unusual partial variants of HPRT-deficiency. HPRT cDNA was amplified by PCR, cloned and analyzed by automated DNA sequence analysis. Twenty-two, unrelated individuals with HPRT deficiency were studied including eight classic Lesch-Nyhan patients and fourteen patients representing the different groups of partial HPRT deficiency. We found a diverse pattern of mutations with point mutations accounting for the majority of abnormal HPRT genes. Nonsense mutations and exon deletions were only found in HPRT cDNA isolated from classic Lesch-Nyhan patients. Mutations associated with partial HPRT-deficiency were frequently located in the amino terminal part of the molecule. A CpG mutational hot spot was identified at the position for Arg-51 in the HPRT protein. Two hyperuricemic patients exhibited unusual splice site mutations: in one this led to the creation of an additional exon in the HPRT gene and in the other part of exon 6 was missing in a subpopulation of the transcripts, producing the effect of a dominant, negative mutation.
Hum Mol Genet 1992 Sep
PMID:Characterization of mutations in phenotypic variants of hypoxanthine phosphoribosyltransferase deficiency. 130 16

Complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, the Lesch-Nyhan syndrome. This disorder has been identified as a candidate for initial attempts at somatic cell gene therapy. We have previously reported the construction of a recombinant herpes simplex virus type 1 (HSV-1) vector containing human hprt cDNA sequences under the regulatory control of the viral thymidine kinase gene (tk) [Palella et al., Mol. Cell. Biol. 8 (1988) 457-460]. Infection of HPRT- cultured rat neuronal cells with these vectors resulted in transient expression of human hprt. In this paper, we report the expression of human hprt mRNA transcripts in the brains of mice infected in vivo with this vector by direct intracranial inoculation. Human hprt transcripts were distinguished from endogenous mouse transcripts by RNase A mapping using riboprobes transcribed from human hprt cDNA. These initial studies demonstrate the transfer and transcription of a human gene in brain cells by direct in vivo infection with recombinant HSV-1 vectors.
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PMID:Expression of human HPRT mRNA in brains of mice infected with a recombinant herpes simplex virus-1 vector. 255 79

The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.
Mol Cell Biol 1988 Jan
PMID:Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells. 282 6

Molecular analysis of an unusual patient with the Lesch-Nyhan syndrome has suggested that the mutation is due to a partial HPRT gene duplication. We now report the cloning and sequencing of the mutant HPRT cDNA which shows the precise duplication of exons 2 and 3. This mutation is the result of an internal duplication of 16-20 kilobases of the gene. The structure of the mutant gene suggests that the duplication was not generated by a single unequal crossing-over event between two normal HPRT alleles. Growth of Epstein-Barr virus-transformed lymphoblasts from this patient in selective medium has permitted isolation of spontaneous HPRT+ revertants of this mutation. The reversion event involves a second major HPRT gene rearrangement where most or all of the duplicated portion of the mutant gene is deleted. The original mutation therefore has the potential for spontaneous somatic reversion. This may explain the relatively mild symptoms of the Lesch-Nyhan syndrome exhibited by this patient.
Somat Cell Mol Genet 1988 May
PMID:Spontaneous reversion of novel Lesch-Nyhan mutation by HPRT gene rearrangement. 283 25

Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 X 10(3) to 5 X 10(4)/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3- to 10-fold by cocultivation of the ecotropic psi 2 and amphotropic PA-12 helper cells. Higher viral titers (8 X 10(5) to 7.5 X 10(6] were obtained when nonproducer NIH 3T3 cells or psi 2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT- (6-thioguanine resistant [6TGr]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418r could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression.
Mol Cell Biol 1987 Feb
PMID:Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow. 346 9

Lesch-Nyhan syndrome is an X-linked recessive disorder caused by molecular defects within the HPRT gene. Deletional forms of this syndrome, most of which are inherited, account for 15% of the cases. In addition, a large percentage of cases are due to de novo point mutations. We have used complementary fluorescence-based PCR assays to analyse disease-causing mutations in three unrelated families: (1) inheritance of dye-labelled PCR products of linked polymorphic loci mapping within and flanking the HPRT gene; (2) dye-labelled exon dosage analysis and (3) automated fluorescence-based DNA sequence analysis. Our results using fluorescent, dye-tagged PCR products show that inheritance of two polymorphic small tandem repeats, HPRTB [AGAT]n, mapping within intron 3 of the HPRT gene, and the CA-repeat at DXS294 can be used to establish linkage to the disease. In addition, we modified a previously described PCR protocol to use fluorescent dye-labelled oligoprimers and an ABI Gene Scanner in order to rapidly quantitate deletional forms of Lesch-Nyhan syndrome. Quantitative PCR analysis of individual exons followed by dosage analysis confirmed a deletion encompassing exon 9. A similar approach was used to confirm a previously described HPRT gene duplication involving exons 2 and 3. In this analysis, we co-amplified the HPRTB [AGAT]n and HUMARA [AGC]n repeats and confirmed increased exon dosage in carriers for the duplication. DNA sequence analysis remains the method of choice for delineating new disease-causing mutations, most of which are non-deletional forms of Lesch-Nyhan syndrome. We have also used a cycle-sequencing strategy employing dye-labelled dideoxy terminators and a laser-activated, fluorescence-emission DNA sequencer in order to define carrier status in 10 family members at risk for Lesch-Nyhan syndrome due to a splice donor mutation in intron 7. Our DNA sequence analyses corroborate small tandem repeat (STR) inheritance patterns in this family. Multiple fluorescence-based strategies should facilitate rapid diagnosis of the various Lesch-Nyhan disease-causing mutations.
Mol Cell Probes 1993 Aug
PMID:Fluorescent approaches to diagnosis of Lesch-Nyhan syndrome and quantitative analysis of carrier status. 823 48

We have used peripheral blood T-lymphocyte cultures to analyze the hprt mutation in two Lesch-Nyhan syndrome males who are cousins and to confirm the carrier status of female members of the family. Both cDNA and genomic DNA sequencing studies show that this patient carries a hitherto undescribed single base deletion in the exon 5 donor splice site sequence (I5: +1, delta G, base number 31635). The largest cDNA product contained all nine hprt exons plus an insertion of 66 bases of intron 5, consistent with the use of a cryptic splice site in intron 5 (aag67/gtaagc). This splicing error would result in a chain terminating codon immediately after exon 5 (I5:2-4, taa) and predicts a polypeptide of 133 amino acids. This loss of the normal splice donor site also results in multiple hprt mRNA species, combining the use of the cryptic splice site in intron 5 and splicing errors involving exons 2-6. In addition to defining a new Lesch-Nyhan mutation (hprtHenryville), these results provide insight into aberrant splicing of hprt mRNA in T-lymphocytes.
Somat Cell Mol Genet 1996 Mar
PMID:Germinal HPRT splice donor site mutation results in multiple RNA splicing products in T-lymphocyte cultures. 878 93

Complete hypoxanthine-guanine phosphoribosyl-transferase (HPRT) deficiency in humans results in the Lesch-Nyhan syndrome which is characterized, among other features, by compulsive self-injurious behavior. HPRT-deficient mice generated using mouse embryonic stem cells exhibit none of the behavioral symptoms associated with the Lesch-Nyhan syndrome. Administration of drugs that inhibit adenine phosphoribosyltransferase (APRT) in HPRT-deficient mice has produced the suggestion that deficiency of APRT in combination with HPRT-deficiency in mice may lead to self-mutilation behavior [C.L. Wu and D.W. Melton (1993) Nature Genet. 3, 235-240]. To test this proposition, we bred HPRT-APRT-deficient mice. Although the doubly-deficient mice excrete adenine and its highly insoluble derivative, 2,8-dihydroxyadenine, which are also associated with human APRT deficiency, additional abnormalities or any self-injurious behavior were not detected. Thus, APRT-HPRT-deficient mice, which are devoid of any purine salvage pathways, show no novel phenotype and are not a model for the behavioral abnormalities associated with the Lesch-Nyhan syndrome as previously suggested.
Hum Mol Genet 1996 Oct
PMID:HPRT-APRT-deficient mice are not a model for lesch-nyhan syndrome. 889 95

We have detected a novel splice variant of the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in two human oocytes and four preimplantation embryos from the 4-cell to the 8-cell stage of development. The novel HPRT transcript lacks exons 4, 5 and 6 of the normal HPRT gene. The same parental origin for the two oocytes and two of the preimplantation embryos, in which the alternatively spliced transcript was detected, might suggest that the alternative splicing is influenced by genetic background. Mutations in the HPRT gene which cause alternative mRNA splicing are implicated in Lesch-Nyhan syndrome. However, the relatively high frequency of detection of this novel HPRT transcript described here (6/109 oocytes and preimplantation embryos) suggests that it is not involved in Lesch-Nyhan syndrome. It is probable that the alternative HPRT transcript is derived from the aberrant splicing of a small percentage of the total mRNA produced from normal HPRT alleles. The presence of this alternative transcript in human preimplantation embryos may complicate an reverse transcription-polymerase chain reaction-based preimplantation diagnosis of Lesch-Nyhan syndrome.
Mol Hum Reprod 1998 Aug
PMID:Detection of a novel splice variant of the hypoxanthine-guanine phosphoribosyl transferase gene in human oocytes and preimplantation embryos: implications for a RT-PCR-based preimplantation diagnosis of Lesch-Nyhan syndrome. 973 36

Lesch-Nyhan disease is a genetic disorder of purine metabolism caused by defective activity of the enzyme hypoxanthine-guanine phosphoribosyl transferase (HPRT), resulting from mutation in the corresponding gene on the long arm of the X chromosome (Xq26). The classical phenotype, which includes spasticity, involuntary movements, developmental disability, and self-injurious behavior, occurs exclusively in males, while heterozygous, carrier females are clinically normal. We analyzed an Argentine family in which there were male and female siblings with clinically identical classic features of Lesch-Nyhan disease. The mother and an older daughter were carriers and had normal phenotypes. We identified the HPRT mutation in the family. It is a C --> T transition at position 508 of the cDNA (c.508 C --> T) that changes the CGA codon for Arg(169) to the TGA stop codon (R169X). The female patient was karyotypically normal and heterozygous for the mutation. She inherited the HPRT mutation from her mother, but she also had unexpected nonrandom inactivation of the paternal X chromosome carrying the normal HPRT gene. This additional genetic alteration is the cause of the clinical expression of disease in this female patient.
Mol Genet Metab 2000 Mar
PMID:An unexpected affected female patient in a classical Lesch-Nyhan family. 1076 82


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