Human Genetics

, Volume 85, Issue 1, pp 111–116 | Cite as

Molecular analyses of a Lesch-Nyhan syndrome mutation (hprtMontreal) by use of T-lymphocyte cultures

  • Thomas R. Skopek
  • Leslie Recio
  • Deborah Simpson
  • Louis Dallaire
  • Serge B. Melancon
  • Helene Ogier
  • J. Patrick O'Neill
  • Michael T. Falta
  • Janice A. Nicklas
  • Richard J. Albertini
Original Investigations

Summary

The frequency of hprt mutants in peripheral blood T-lymphocytes of two putative Lesch-Nyhan individuals and their parents was determined by a cell cloning assay to quantify the frequency of thioguanine-resistant mutants. The results confirmed the Lesch-Nyhan diagnosis and demonstrated that the mother has an elevated mutant frequency consistent with being heterozygous for an hprt mutation. Mass cultures of T-lymphocytes from both the children and their mother, as well as cultures of hprt mutant clones from the mother, were employed as sources of mRNA for cDNA sequence analysis. These hprt mutants show a single base substitution (T→C transition) at position 170 (exon 3). The predicted amino acid change is the substitution of threonine for methionine56. We have designated this new Lesch-Nyhan mutation hprtMontreal. The use of T-lymphocyte cultures allows rapid sequence analyses of hprt mutations, as well as family studies to define the origin of a particular mutation.

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References

  1. Albertini RJ, DeMars R (1974) Mosaicism of peripheral blood lymphocyte populations in females heterozygous for the Lesch-Nyhan mutation. Biomed Genet 11:397–411Google Scholar
  2. Albertini RJ, Castle KL, Borcherding WR (1982) T-cell cloning to detect the mutant 6-thioguanine resistant lymphocytes present in human peripheral blood. Proc Natl Acad Sci USA 79:6617–6621Google Scholar
  3. Albertini RJ, O'Neill JP, Nicklas JA, Heintz NH, Kelleher PC (1985) Alterations of the hprt gene in human in vivo derived 6 thioguanine-resistant T-lymphocytes. Nature 316:369–371Google Scholar
  4. Albertini RJ, Sullivan LM, Berman JK, Greene CJ, Stewart JA, Silveira JM, O'Neill JP (1988) Mutagenicity monitoring in humans by autoradiographic assay for mutant T-lymphocytes. Mutat Res 204:481–492Google Scholar
  5. Argos P, Hanei M, Wilson JM, Kelly WN (1983) A possible nucleotide-binding domain in the tertiary fold of phosphoribosyl transferases. J Biol Chem 258:6450–6457Google Scholar
  6. Bradley JE, Bishop GA, St John T, Frelinger JA (1988) A simple, rapid method for the purification of poly A+ RNA. Biotechniques 6:115–116Google Scholar
  7. Chou PY, Fasman GD (1978) Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol 47:45–148Google Scholar
  8. Davidson BL, Chen S-J, Wilson JM, Kelley WN, Palella TD (1988a) Hypoxanthine-guanine phosphoribosyltransferase: genetic evidence for identical mutations in two partially deficient subjects. J Clin Invest 82:2164–2167Google Scholar
  9. Davidson BL, Palella TD, Kelley WN (1988b) Human hypoxanthine-guanine phosphoribosyltransferase: a single nucleotide substitution in cDNA clones isolated from a patient with Lesch-Nyhan syndrome (HPRTMidland). Gene 68:85–91Google Scholar
  10. Davidson BL, Pashmforoush M, Kelley WN, Palella TD (1988c) Genetic basis of hypoxanthine guanine phosphoribosyltransferase deficiency in a patient with the Lesch-Nyhan syndrome (HPRTFlint). Gene 63:331–336Google Scholar
  11. Davidson BL, Pashmforoush M, Kelley WN, Palella TD (1989a) Human hypoxanthine guanine phosphoribosyltransferase deficiency. The molecular defect in a patient with gout (HPRTAshville). J Biol Chem 264:520–525Google Scholar
  12. Davidson BL, Tarle SA, Palella TD, Kelley WN (1989b) Molecular basis of hypoxanthine guanine phosphoribosyltransferase deficiency in ten subjects determined by direct sequencing of amplified transcripts. J Clin Invest 84:342–346Google Scholar
  13. Francke U, Bakay B, Nyhan WL (1973) Detection of heterozygous carriers of the Lesch-Nyhan syndrome by electrophoresis of hairroot lysates. J Pediatr 82:472–478Google Scholar
  14. Fujimori S, Hidaka Y, Davidson BL, Palella TD, Kelley WN (1988) Identification of a single nucleotide change in a mutant gene for hypoxanthine-guanine phosphoribosyltransferase (HPRTAnn Arbor). Hum Genet 79:39–43Google Scholar
  15. Fujimori S, Davidson BL, Kelley WN, Palella TR (1989) Identification of a single nucleotide change in the hypoxanthine guanine phosphoribosyltransferase gene (HPRTYale) responsible for Lesch-Nyhan syndrome. J Clin Invest 83:11–13Google Scholar
  16. Gibbs RA, Caskey CT (1987) Identification and localization of mutations at the Lesch-Nyhan locus by ribonuclease A cleavage. Science 236:303–305Google Scholar
  17. Gibbs RA, Nguyen PN, McBride LJ, Koepf SM, Caskey CT (1989) Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA. Proc Natl Acad Sci USA 86:1919–1923Google Scholar
  18. Hopp TP, Woods KR (1981) Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci USA 78:3824–3828Google Scholar
  19. Jolly DJ, Okayama H, Berg P, Esty AC, Filipula D, Bohlen P, Johnson GG, Shively JE, Hunkapillar T, Freedman T (1983) Isolation and characterization of a full length cDNA for human hypoxanthine phosphoribosyltransferase. Proc Natl Acad Sci USA 80:477–481Google Scholar
  20. Kelley W, Greene M, Rosenbloom F, Henderson J, Seegmiller J (1969) Hypoxanthine-guanine phosphoribosyltransferase deficiency in gout. Ann Intern Med 70:155–206Google Scholar
  21. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132Google Scholar
  22. Lesch M, Nyhan W (1964) A familiar disorder of uric acid metabolism and central nervous system function. Am J Med 36:561–570Google Scholar
  23. Migeon B, DerKaloustian VM, Nyhan WL, Young WJ, Childs B (1968) X-linked HG-PRT deficiency: heterozygote has two clonal populations. Science 160:425–428Google Scholar
  24. Miller JH (1983) Mutational specificity in bacteria. Annu Rev Genet 17:215–238Google Scholar
  25. Nicklas JA, Hunter TC, Sullivan LM, Berman JK, O'Neill JP, Albertini RJ (1987) Molecular analyses of in vivo hprt mutations in human T-lymphocytes. I. Studies of low frequency “spontaneous” mutants by Southern blots. Mutagenesis 2:341–347Google Scholar
  26. Nicklas JA, O'Neill JP, Sullivan LM, Hunter TC, Allegretta M, Chastenay BF, Libbus BL, Albertini RJ (1988) Molecular analysis of in vivo hprt mutations in human T-lymphocytes: demonstration of a clonal amplification of hprt mutant T-lymphocytes in vivo. Environ Mol Mutagen 12:271–284Google Scholar
  27. O'Neill JP, McGinniss MJ, Berman JK, Sullivan LM, Nicklas JA, Albertini RJ (1987) Refinement of a T-lymphocyte cloning assay to quantify the in vivo thioguanine resistant mutant frequency in humans. Mutagenesis 2:87–94Google Scholar
  28. O'Neill JP, Sullivan LM, Booker JK, Pornelos BS, Falta MT, Greene CJ, Albertini RJ (1989) Longitudinal study of the in vivo hprt mutant frequency in human T-lymphocytes as deterined by a cell cloning assay. Environ Mol Mutagen 13:289–293Google Scholar
  29. Rosenbloom FM, Kelly WN, Henderson JF, Seegmiller JE (1967a) Lyon hypothesis and X-linked disease. Lancet II:305–309Google Scholar
  30. Rosenbloom F, Kelley W, Miller J, Henderson J, Seegmiller J (1967b) Inherited disorder of purine metabolism: correlation between central nervous system dysfunction and biochemical defects. JAMA 202:103–106Google Scholar
  31. Seegmiller J, Rosenbloom F, Kelley W (1967) Enzyme defect associated with a sex linked human neurological disorder and excessive purine synthesis. Science 155:1682–1684Google Scholar
  32. Silverman J, Kelley WN, Patella TD (1987) Genetic analysis of human hypoxanthine-guanine phosphoribosyltransferase deficiency. Enzyme 38:36–44Google Scholar
  33. Simpson D, Crosby RM, Skopek TR (1988) A method for specific cloning and sequencing of human hprt cDNA for mutation analyses. Biochem Biophys Res Commun 151:487–492Google Scholar
  34. Sinnett D, Lavergne L, Melancon SB, Dallaire L, Potier M, Labuda D (1988) Lesch-Nyhan syndrome: molecular investigation of three French Canadian families using a hypoxanthineguanine phosphoribosyltransferase cDNA probe. Hum Genet 81:4–8Google Scholar
  35. Strauss GH, Allen EF, Albertini RJ (1980) An enumerative assay of purine analogue resistant lymphocytes in women heterozygous for the Lesch-Nyhan mutation. Biomed Genet 18:529–547Google Scholar
  36. Wilson J, Kobayashi R, Fox I, Kelley W (1983) Human hypoxanthine guanine phosphoribosyltransferase: molecular abnormality in a mutant form of the enzyme (HPRT in Toronto) J Biol Chem 258:6458–6460Google Scholar
  37. Wilson J, Stout J, Palella T, Davidson B, Kelley W, Caskey C (1986) A molecular survey of hypoxanthine-guanine phosphoribosyltransferase deficiency in man. J Clin Invest 77:188–195Google Scholar
  38. Yang TP, Patel PI, Chinault AC, Stout JT, Jackson LG, Hildebrand BM, Caskey CT (1984) Molecular evidence for new mutation at the hprt locus in Lesch-Nyhan patients. Nature 310:412–414Google Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • Thomas R. Skopek
    • 1
  • Leslie Recio
    • 1
  • Deborah Simpson
    • 1
  • Louis Dallaire
    • 2
  • Serge B. Melancon
    • 2
  • Helene Ogier
    • 2
  • J. Patrick O'Neill
    • 3
  • Michael T. Falta
    • 3
  • Janice A. Nicklas
    • 3
  • Richard J. Albertini
    • 3
  1. 1.Chemical Industry Institute of ToxicologyUSA
  2. 2.Hôpital Sainte JustineMontrealCanada
  3. 3.Genetics Laboratory, University of VermontBurlingtonUSA

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