Identification of Mutations Leading to the Lesch-Nyhan Syndrome by Automated Direct DNA Sequencing of in vitro Amplified cDNA

• Published in: Proceedings of the National Academy of Sciences - PNAS 1989-03, Vol.86 (6), p.1919-1923
• Main Authors: Gibbs, Richard A., Nguyen, Phi-Nga, McBride, Lincoln J., Koepf, Sandy M., Caskey, C. Thomas
• Format: Article
• Language: English
• Subjects: 550401 - Genetics- Tracer Techniques; Alleles; ANIMAL CELLS; ANIMALS; Autoanalysis; AUTOMATION; Base Sequence; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; Biological and medical sciences; BIOLOGICAL MATERIALS; BLOOD; BLOOD CELLS; BODY FLUIDS; CHROMOSOMES; Classical genetics, quantitative genetics, hybrids; Complementary DNA; CONNECTIVE TISSUE CELLS; DAYS LIVING RADIOISOTOPES; DIAGNOSIS; DISEASES; DNA; DNA - genetics; DNA sequence analysis; DNA SEQUENCING; DNA-Directed DNA Polymerase; ENZYMES; Exons; Fundamental and applied biological sciences. Psychology; Gels; GENE AMPLIFICATION; GENE MUTATIONS; Genetic mutation; Genetics of eukaryotes. Biological and molecular evolution; GLYCOSYL TRANSFERASES; HEREDITARY DISEASES; HETEROCHROMOSOMES; Human; Humans; HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE; Hypoxanthine Phosphoribosyltransferase - genetics; IN VITRO; ISOTOPES; Lesch Nyhan syndrome; Lesch-Nyhan Syndrome - genetics; LEUKOCYTES; LIGHT NUCLEI; LYMPHOCYTES; MAMMALS; MAN; MATERIALS; Mutation; MUTATIONS; NUCLEI; Nucleic Acid Hybridization; NUCLEIC ACIDS; ODD-ODD NUCLEI; Oligonucleotide Probes; Oligonucleotides; ORGANIC COMPOUNDS; Pedigree; PENTOSYL TRANSFERASES; PHOSPHORUS 32; PHOSPHORUS ISOTOPES; Polymerase chain reaction; PRIMATES; RADIOISOTOPES; RECOMBINANT DNA; RNA Splicing; Sequencing; Software; SOMATIC CELLS; STRUCTURAL CHEMICAL ANALYSIS; TRANSFERASES; VERTEBRATES; X CHROMOSOME
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.86.6.1919

The Lesch-Nyhan (LN) syndrome is a severe X chromosome-linked disease that results from a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). The mutations leading to the disease are heterogeneous and frequently arise as de novo events. We have identified nucleotide alterations in 15 independently arising HPRT-deficiency cases by direct DNA sequencing of in vitro amplified HPRT cDNA. We also demonstrate that the direct DNA sequence analysis can be automated, further simplifying the detection of new mutations at this locus. The mutations include DNA base substitutions, small DNA deletions, a single DNA base insertion, and errors in RNA splicing. The application of these procedures allows DNA diagnosis and carrier identification by the direct detection of the mutant alleles within individual families affected by LN.