thioguanine-anhydrous and Lesch-Nyhan-Syndrome

The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion ("ScanDel"). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder. Altogether, we programmed 4,342 overlapping 1 and 2 kb deletions that tiled 206 kb centered on HPRT1 (including 87 kb upstream and 79 kb downstream) with median 27-fold redundancy per base. We functionally assayed programmed deletions in parallel by selecting for loss of HPRT function with 6-thioguanine. As expected, sequencing gRNA pairs before and after selection confirmed that all HPRT1 exons are needed. However, HPRT1 function was robust to deletion of any intergenic or deeply intronic non-coding region, indicating that proximal regulatory sequences are sufficient for HPRT1 expression. Although our screen did identify the disruption of exon-proximal non-coding sequences (e.g., the promoter) as functionally consequential, long-read sequencing revealed that this signal was driven by rare, imprecise deletions that extended into exons. Our results suggest that no singular distal regulatory element is required for HPRT1 expression and that distal mutations are unlikely to contribute substantially to Lesch-Nyhan syndrome burden. Further application of ScanDel could shed light on the role of regulatory mutations in disease at other loci while also facilitating a deeper understanding of endogenous gene regulation.

Topics: Cell Line; CRISPR-Cas Systems; Gene Expression Regulation; HEK293 Cells; Humans; Hypoxanthine Phosphoribosyltransferase; Lesch-Nyhan Syndrome; Regulatory Sequences, Nucleic Acid; RNA, Guide, Kinetoplastida; Sequence Deletion; Thioguanine

Hypoxanthine phosphoribosyl transferase (HPRT, also known as HGPRT) is an often-used genetic marker in eukaryotic cells. The gene is conserved from bacteria to human, with retained catalytic activity, although substrate specificity may have changed, and the enzyme is essential in malaria-causing protozoans. Inherited mutations in the human HPRT1 gene result in three different phenotypes: Lesch-Nyhan syndrome (LNS or LND), LND variants, and HPRT-related hyperuricemia (HRH). In cultured cells, loss of HPRT activity gives rise to 6-thioguanine (6-TG) resistance. In general, cells from LND patients are also 6-TG resistant, whereas cells from HRH patients are not, with some interesting exceptions. Using modeling methods, we have studied the correlation between the mutable and nonmutated amino acid residues on one hand, and sequence conservation and predicted phenotypic effects on the other hand. Our results demonstrate that most of the mutations are explainable by the predicted effect on protein structure and function. They are also consistent with sequence conservation. Moreover, the mutational profiles of TG-resistant cells and LND overlap to a great extent, while most of the mutations in HRH are unique to that condition. We have also noticed a strong correlation between mutations in the tetramer interfaces and observed phenotypes, suggesting a functional role for a tetramer transition during catalysis.

Topics: Amino Acid Sequence; Drug Resistance; Germ-Line Mutation; Humans; Hyperuricemia; Hypoxanthine Phosphoribosyltransferase; Lesch-Nyhan Syndrome; Molecular Sequence Data; Mutation; Phenotype; Protein Conformation; Structure-Activity Relationship; Thioguanine

Lesch-Nyhan syndrome is caused by a severe genetic deficiency of hypoxanthine phosphoribosyltransferase (HPRT) and is characterized by central nervous system disorders, gout, and in some cases, macrocytic anemia. Women heterozygous for HPRT deficiency are healthy but their somatic cells are mosaic for enzyme deficiency owing to random inactivation of the X chromosome. Frequencies of red blood cells and T cells deficient in HPRT are significantly lower than the expected 50% in heterozygotes, suggesting that HPRT-negative blood cells are selected against in heterozygotes. To determine at which stage of hematopoiesis such selection occurs, we determined the frequencies of HPRT-negative T, B and erythroid precursor cells in three heterozygotes. Since the cloning efficiencies of T and B cells and colony forming efficiency of burst-forming unit erythroid (BFU-E) for sample from Lesch-Nyhan patients were similar to those of normal cells, HPRT deficiency does not seem to render the differentiated cells less efficient for proliferation. However, the frequencies of HPRT-negative T and B cells, and BFU-E were all less than 10% in each of the three heterozygotes. Although the frequencies of HPRT-negative cells showed tenfold variations between the heterozygotes, each heterozygote had similar frequencies of HPRT-negative cells in the three cell types. These results suggest that HPRT is important at early stages of hematopoiesis, but less so after the cells have differentiated into T cells, B cells and erythroid precursor cells.

Topics: Adult; B-Lymphocytes; Cell Line; Clone Cells; Erythrocytes; Female; Genetic Carrier Screening; Hematopoiesis; Hematopoietic Stem Cells; Heterozygote; Humans; Hypoxanthine Phosphoribosyltransferase; Lesch-Nyhan Syndrome; Organ Specificity; T-Lymphocytes; Thioguanine; X Chromosome

Topics: Azaserine; Cells, Cultured; Genetic Carrier Screening; Humans; Hypoxanthine; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Kinetics; Lesch-Nyhan Syndrome; Lymphocyte Activation; Lymphocytes; Male; Reference Values; Thioguanine

The feasibility of using RNA synthesis in freshly isolated, human peripheral blood lymphocytes to detect 6-thioguanine (TG)- and 8-azaguanine (AG)-resistant variants in an autoradiographic assay similar to that of Strauss and Albertini (1979) has been evaluated. In phytohemagglutinin (PHA)-stimulated cultures RNA synthesis and HPRT activity began well in advance of DNA synthesis and increased in parallel during the first 44 h of culture. Introduction of TG or AG with PHA at the beginning of culture completely inhibited DNA synthesis during the first 44 h and reduced RNA synthesis to low levels within 24 h. When TG or AG was added after cells had been in culture for 38 h, DNA synthesis was reduced quickly while RNA synthesis was inhibited more slowly. An autoradiographic assay is described in which freshly isolated lymphocytes are cultured with PHA for 24 h, with or without TG or AG, then labeled with [3H]uridine for 1 h. TG-resistant and AG-resistant variant frequencies for 2 normal individuals and a Lesch-Nyhan individual were determined with this assay. The variant frequencies for the normal individuals ranged from 0.46 to 10.6 X 10(-5) depending upon the selective conditions used. All the Lesch-Nyhan cells were resistant to 0.2 microM-2 mM AG; some were sensitive to 0.2 mM TG and most were sensitive to 2.0 mM TG.

Topics: Autoradiography; Azaguanine; Drug Resistance; Genetic Variation; Humans; Hypoxanthine Phosphoribosyltransferase; In Vitro Techniques; Lesch-Nyhan Syndrome; Lymphocytes; Phytohemagglutinins; RNA; Thioguanine

Peripheral T cells from 3 Lesch-Nyhan patients, 3 normal subjects, and 3 brothers with hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency but without Lesch-Nyhan syndrome (so-called partial deficiency) have been analyzed. Although these brothers contained HGPRT activities neither in the hemolysates nor in the T cell extracts at levels detectable by the regular radioenzyme assay, the enzyme deficiency had not caused any typical neurological symptoms of the Lesch-Nyhan syndrome. Although the T cells from these brothers were at least 10-fold more resistant to 6-thioguanine than normal T cells, they were more than 30-fold less resistant than the T cells from 3 Lesch-Nyhan patients indicating that there is a clear difference in the severity of the enzyme deficiency between the brothers and the Lesch-Nyhan patients. These data indicate that the long-term T cell culture in the medium containing a purine analog whose toxicity depends on a salvaging enzyme is useful for evaluating the severity of the enzyme deficiency in viable cells.

Topics: Adolescent; Cells, Cultured; Female; Genetic Carrier Screening; Humans; Hypoxanthine Phosphoribosyltransferase; Lesch-Nyhan Syndrome; Male; T-Lymphocytes; Thioguanine

A human Lesch-Nyhan (hereditary, severe hypoxanthine-guanine phosphoribosyltransferase (HPR transferase) deficiency) B-lymphoblast line was infected with an amphotropic retroviral vector containing human HPR transferase cDNA under transcriptional control of viral long terminal repeat sequences. Of 17 clones isolated, 12 integration groups were defined by analysis of restriction enzyme digests of their genomic DNA with HPR transferase and viral long terminal repeat probes. These groups had HPR transferase activity restored to levels of 4 to 23% of normal values. Aberrant metabolic parameters associated with severe deficiency of HPR transferase activity, i.e. elevated rates of purine excretion, increased accumulation of hypoxanthine, elevated 5-phosphoribosyl-1-pyrophosphate contents, altered nucleoside triphosphate pools, resistance to toxic effects of 6-thioguanine, were partially to nearly completely corrected; the degree of correction generally corresponded to the degree of restoration of HPR transferase activity. The integration of the HPR transferase gene was found to be variably stable during 9 months of culture of the virally transformed lymphoblasts under nonselective conditions. The HPR transferase gene-infected lines reverted to resistance to 20 microM 6-thioguanine, i.e. severe HPR transferase deficiency, at frequencies of 10(-6) to in excess of 10(-5) per generation. The reversions were accompanied by either a loss or rearrangement of the integrated HPR transferase sequences or by retention of the sequences in an unaltered form.

Topics: Animals; B-Lymphocytes; DNA; Gout; Humans; Hypoxanthine Phosphoribosyltransferase; Lesch-Nyhan Syndrome; Male; Mice; Phenotype; Phosphoribosyl Pyrophosphate; Purines; Repetitive Sequences, Nucleic Acid; Retroviridae; Thioguanine; Transcription, Genetic; Transfection

Thioguanine-resistant T lymphoblast populations were selectively amplified using T cell growth factor in the cultures of peripheral blood T cells from four Lesch-Nyhan heterozygotes. Although Lesch-Nyhan T lymphoblasts were all thioguanine-resistant, none of the cultures from 13 control subjects yielded the growth of such defective cell populations. These data provide direct evidence for the existence of a small percentage (5%-40%) of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient T cells in the heterozygotes, but not in normal individuals. Conversely, culture of the T lymphoblasts with azaserine plus hypoxanthine permitted the growth of the other part of the cell population that was enzyme positive. The low percentages of HGPRT-negative cells among T cells in heterozygotes suggest that the presence of this enzyme is beneficial for differentiation of lymphocytes of T cell linkage. Considering the ease and the reliability, culture of the peripheral T cells with thioguanine and T cell growth factor is very likely of practical use for detecting Lesch-Nyhan syndrome carriers among predisposed females.

Topics: Adolescent; Cell Division; Cells, Cultured; Child; Culture Media; Female; Genetic Carrier Screening; Humans; Interleukin-2; Lesch-Nyhan Syndrome; Lymphocyte Activation; Male; T-Lymphocytes; Thioguanine

Topics: Cell Division; Cell Line; Cell Transformation, Neoplastic; Drug Resistance; Humans; Lesch-Nyhan Syndrome; Mutation; Skin Neoplasms; Skin Physiological Phenomena; Sodium-Potassium-Exchanging ATPase; Thioguanine

Topics: Adenosine; Bromodeoxycytidine; Cells, Cultured; Deoxycytidine; DNA, Viral; Herpes Simplex; Humans; Lesch-Nyhan Syndrome; Orotic Acid; Thioguanine; Ultraviolet Rays; Virus Replication; Xeroderma Pigmentosum

Using a limiting dilution technique, we found that the frequency of thioguanine resistant (TGR) lymphocyte clones was less than 5.0 X 10(-5) in 14 normal individuals, between 9.0 X 10(-3) and 8.9 X 10(-2) in seven heterozygotes for Lesch-Nyhan syndrome, and 0.88 and 0.87 in two hemizygotes. TGR clones from heterozygotes were expanded and had the hemizygote phenotype as evidenced by low hypoxanthine incorporation and severely deficient hypoxanthine-guanine-phosphoribosyl-transferase activity. Enumeration of TGR lymphocyte clones provides a simple technique for detection of heterozygosity for Lesch-Nyhan syndrome. A similar approach using lymphocyte cloning may be suitable for detection of the carrier state for other X-linked disorders.

Topics: Clone Cells; Drug Resistance; Female; Genetic Carrier Screening; Humans; Lesch-Nyhan Syndrome; Lymphocytes; Male; Thioguanine

In females heterozygous for the Lesch-Nyhan (LN) mutation, there is mosaicism of peripheral blood lymphocytes (PBLs) with regard to sensitivity to 6-thioguanine (TG) inhibition of tritiated thymidine ([3H]Tdr) incorporation following phytohemagglutinin (PHA) stimulation. That there are two populations of PBLs, normal and mutant (LN-like), has been demonstrated by an autoradiographic enumerative assay. A single three-generation family containing six potentially heterozygous females was studied. Five of the six were mosaics with frequencies of TG-resistant (TGr) PBLs rangling from 1.4 x 10(-3) to 4.2 x 10(-3) when tested at 2 x 10(-4) M TG. The median frequency of TGr PBLs in 63 healthy non-LN individuals between the ages of 11 and 75 years was found to be 1.1 x 10(-4) (mean 1.3 x 10(-4)) (10th and 90th percentiles--6.1 x 10(-5) and 2.1 x 10(-4)) and was not age related. The sixth potentially heterozygous female in the current family had a TGr PBL frequency of 1.9 x 10(-4). In the five females with elevated TGr PBL frequencies, TGr skin fibroblasts with frequencies ranging from 26% to 100% of the sample tested were found; in the female with the normal TGr PBL frequency, no TGr skin fibroblasts were found. The former group was considered to be LN heterozygous. Four of the five had been previously diagnosed as such. The latter individual is considered to be genotypically normal. Females who are heterozygotes for the LN mutation have two populations of PBLs.

Topics: Adolescent; Adult; Aged; Autoradiography; Child; Drug Resistance; Female; Fibroblasts; Genetic Carrier Screening; Humans; Hypoxanthine Phosphoribosyltransferase; Lesch-Nyhan Syndrome; Lymphocyte Activation; Lymphocytes; Male; Middle Aged; Mosaicism; Pedigree; Phytohemagglutinins; Thioguanine; Thymidine

An autoradiographic method to enumerate variant 6-thiogunanine-resistant (TGr) peripheral blood lymphocytes (PBLs) that occur in vivo in man is described. Variant cells are detected in PBL cultures stimulated to tritiated thymidine (3HTdr) incorporation in vitro with phytohemagglutinin (PHA) in the presence of TG. Cells with the naturally-occurring Lesch--Nyhan (LN) mutation served as prototype-variant cells. PBLs from a LN hemizygous male were found to be resistant to TG inhibition of PHA-stimulated 3HTdr in corporation in vitro while a LN heterozygous female was found to be a mosaic with 2/1000 PBLs resistant to 2 X 10(-4) M TG. Experiments with artificial mixtures of LN and normal PBLs showed that the LN cells were virtually all detectable even when present in low frequency (10(-5)). TGr PBLs were found in healthy non-LN individuals at median frequencies of 1.0 X 10(-4) and 1.1 X 10(-4) when determined at 2 X 10(-3) M TG and 2 X 10(-4) M TG respectively. Their frequencies were not age-related. TGr PBL-variant frequencies (Vf's) were determined in 47 cancer patients who were being treated with cytotoxic agents that are known to be mutagens. The median TGr PBL Vf determined at 2 X 10(-3) M TG in cancer patients was 2.2 X 10(-4) while, when determined at 2 X 10(-4) M TG, it was 8.5 X 10(-4). The distribution of Vf's for the treated cancer-patient group differed from that for the normal control group in that more than half of the treated cancer patients had TGr PBL Vf's greater than the highest seen for controls. Unlike those of the normal controls, the TGr PBL Vf's of treated cancer patients differed if determined at 2 X 10(-3) M TG and 2 X 10(-4) M TG, a behavior that suggested partial resistance and mimicked that seen with LN TGr PBLs. PBLs resistant to 2,6-diaminopurine (DAPr) were not found in two individuals, although the TGr PBL Vf was elevated in one. TGr PBL Vf's were greatly elevated under conditions of in vivo selection in patients receiving purine-analogue immunosuppressive therapy. The TGr PBL enumerative assay system is presented as one of potential value to detect somatic cell mutations occurring in vivo in man.

Topics: Cytological Techniques; Drug Resistance; Female; Genetic Techniques; Humans; Lesch-Nyhan Syndrome; Lymphocytes; Male; Mutation; Neoplasms; Phenotype; Thioguanine

Purine metabolism has been examined in a clonal line of mouse neuroblastoma cells resistant to the cytotoxic effects of 6-thioguanine. Comparative studies in the resistant and parental lines indicate that the former cells have less than 1% of normal hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) activity. The activities of other enzymes important in the de novo and salvage pathways of purine biosynthesis were not significantly different in the two lines. Hypoxanthine phosphoribosyltransferase deficiency in this neuroblastoma line was associated with increased intracellular concentrations of 5-phosphoribosyl-1-pyrophosphate, an increased rate of purine biosynthesis de novo, and failure to incorporate hypoxanthine, but not adenine, into nucleotides. There are essentially the same alterations in purine metabolism that occur in hypoxanthine phosphoribosyltransferase-deficient fibroblasts or lymphoblasts derived from individuals with the Lesch-Nyhan syndrome. Clonal lines of hypoxanthine phosphoribosyltransferase-deficient neuroblastoma cells may therefore be of use in elucidating the mechanisms by which the enzyme defect leads to the neurologic dysfunction seen in children with this disease.

Topics: Adenine; Animals; Carbon Radioisotopes; Clone Cells; Drug Resistance; Glutamine; Humans; Hypoxanthines; Lesch-Nyhan Syndrome; Mice; Neuroblastoma; Pentosyltransferases; Purine Nucleotides; Purines; Thioguanine

The presence of selectable genetic markers in long-term human lymphoblast cultures would facilitate cell hybridization experiments on the biosynthesis of immunoglobulins, as well as other studies. This work reports the induction with ethylmethane sulfonate of 6-thioguanine - resistant, phosphoribosyltransferase - deficient mutants in a lymphoblast line from a patient with infectious mononucleosis. These cells were unusually sensitive, with a D(0) value of 28 mug of ethylmethane sulfonate per ml; the sensitivity curve followed a biphasic pattern suggesting the presence of 3% resistant cells. Ethylmethane sulfonate increased the frequency of mutants resistant to 6-thioguanine over 100-fold, to about 2 x 10(-4); nitrosoguanidine was less effective. Almost all the mutants contained considerably less than 1% of the hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) activity of wild-type cells. The mutation did not appear to result from loss of an X chromosome.

Topics: Alkanes; Cell Line; Cells, Cultured; Chromosome Aberrations; Female; Gene Frequency; Genetic Code; Humans; Hybridization, Genetic; Infectious Mononucleosis; Inosine Nucleotides; Lesch-Nyhan Syndrome; Lymphocytes; Mutation; Nitrosoguanidines; Pentosephosphates; Pentosyltransferases; Selection, Genetic; Sulfonic Acids; Thioguanine

Cultured fibroblasts established from skin biopsies from patients with the Lesch-Nyhan syndrome are deficient in hypoxanthine-guanine phosphoribosyl-transferase (EC 2.4.2.8) activity. This deficiency makes possible the use of chemicals that select either for or against deficient variants in cultured fibroblasts. Two-way selection has been achieved by the use of 6-thioguanine, which selects for the deficient mutant, and azaserine, which selects to some extent for the normal allele in mixed cultures, as well as in cultures from heterozygotes. Theoretical considerations predict that the phenomenon of metabolic cooperation would tend to reinforce the former and to weaken the latter type of selection, and this is in accordance with the experimental findings.

Topics: Autoradiography; Azaserine; Cell Line; Culture Techniques; Fibroblasts; Genetics, Medical; Guanine Nucleotides; Heterozygote; Humans; Hypoxanthines; Lesch-Nyhan Syndrome; Mutation; Pentosephosphates; Selection, Genetic; Skin; Thioguanine; Time Factors; Transferases; Tritium