title: Fluorescent approaches to diagnosis of Lesch-Nyhan syndrome and quantitative analysis of carrier status.
abstract: 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.
authors.affiliation: Applied Biosystems, Inc., Foster City, CA.
authors.forename: E S
authors.initials: ES
authors.lastname: Mansfield
authors.forename: A
authors.initials: A
authors.lastname: Blasband
authors.forename: M N
authors.initials: MN
authors.lastname: Kronick
authors.forename: L
authors.initials: L
authors.lastname: Wrabetz
authors.forename: P
authors.initials: P
authors.lastname: Kaplan
authors.forename: E
authors.initials: E
authors.lastname: Rappaport
authors.forename: M
authors.initials: M
authors.lastname: Sartore
authors.forename: T
authors.initials: T
authors.lastname: Parrella
authors.forename: S
authors.initials: S
authors.lastname: Surrey
authors.forename: P
authors.initials: P
authors.lastname: Fortina
mesh_terms: D000293:Adolescent; D000328:Adult; D001483:Base Sequence; D017931:DNA Primers; D005260:Female; D008040:Genetic Linkage; D006579:Heterozygote; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D007438:Introns / Q000235:genetics; D007926:Lesch-Nyhan Syndrome / Q000175:diagnosis* / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D005810:Multigene Family; D010375:Pedigree; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
chemical_list: D017931:DNA Primers; D007041:Hypoxanthine Phosphoribosyltransferase