3, No. 3 523. Trinucleotide repeat polymorphism at the PKLR locus. C.Lenzner ... Forward: (5'-5n3CAlF) 5' TTC AGT TTC TCT CGG TGT TCC T 3'. Reverse: ...
Human Molecular Genetics, 1994, Vol. 3, No. 3 523
Two dinucleotide repeat polymorphisms at the DMD locus
Trinucleotide repeat polymorphism at the PKLR locus C.Lenzner, G.Jacobasch, A.Reis\ B.Thiele and P.Nurnberg* Institut fur Medmnische Genetk and Institut fur Biochemie, Hurnboldt-UniversitSt Berlin (Chante), 10098 Bertn and ' Institut fur Humangenetik, Freie Unrversrtat, Heubnerweg 6, 14059 Berlin, Germany
Source: Two (CA)n repeat-containing Sau3A fragments were identified in a pUC18 library of yeast strain AB1380 containing DMD YACs 5'-5 and 3'-19 (1) respectively, by hybridization with poly(dC-dA).poly(dG-dT) (Pharmacia). The recombinants were sequenced using an ABI sequencer and flanking primers were assigned using the programme PRIMER.
Source/Description: The cDNA sequence of the human pyruvate kinase L gene has been published (1). This was used to design primers for amplification of intronic regions. A polymorphic ATT repeat was identified in intron 11.
Primer Sequences: 5'-5n3 ex DMD YAC 5'-5 (EMBL accession number X75801).
Primer Sequences: JHATT: 5'-GGCTGGTTCTCGTTACAG-3' JRS1 : 5'-CTGAGGTCAGGAGTTTGAG-3'
The predicted length of the PCR product was around 330 bp.
Forward: (5'-5n3CAlF) 5' TTC AGT TTC TCT CGG TGT TCC T 3' Reverse: (5'-5n3CAlR) 5' TAC ACC TGC ACA TGT GAT GAA A 3' Repeat sequence: T
Allele Frequency: Estimated from 82 chromosomes of unrelated individuals.
Allele Frequency: Estimated from 73 X chromosomes of unrelated individuals. Observed heterozygosity = 0.76.
Allele Al A2 A3
Allele Al A2 A3 A4 A5
(bp) 116 114 112 110 108
Frequency 0.11 0.29 0.37 0.06 0.03
AUele A6 A7
A8 A9
(bp) 106 104 100 96
Frequency 0.03 0.05 0.04 0.03
Primer sequences: 3'-19n8 ex DMD YAC 3'-19 (EMBL accession number X75580). Forward: (3'-19n8CAF) 5' AGC CCC ATT CTG TAC ATC AAA T 3' Reverse: (3'-19n8CAR) 5' AAC GAC TTC CCC CAC TCT GT 3' Repeat sequence: (AC)| 8
Allele Frequency: Estimated from 69 X chromosomes of unrelated individuals. Observed heterozygosity = 0.58. Allele Al A2 A3
(bp) 150 148 146
Frequency 0.13 0.59 0.23
Allele A4
(bp) 144
A5
140
Frequency 0.03 0.01
PCR Conditions: Amplifications were performed in a final volume of 10 /A containing 50 ng genomic DNA, 0.2 /tM of each primer, 1.5 mM MgCl2, 10 mM Tris-HCl pH 8.4, 50 mM KC1, 200 nM of each dNTP, 0.5 unit of Taq DNA polymerase (Boehringer-Mannheim) and 0.02 /tM of each forward primer that had been 5' end-labelled with gamma-32P-ATP (3000 Ci/mmole; Amersham). 2 minutes at 94 °C; 32 cycles at 61 °C (for YAC 5'-5 repeat) or 63°C (YAC 3'-19 repeat) for 30 seconds; 72°C for 30 seconds; 94°C for 1 minute using an Hybaid Omnigene thermal cycler. Products were separated by electrophoresis in an 8 M urea, 6% polyacrylamide denaturing gel and analysed by autoradiography. Acknowledgements: Supported by the Matamata Muscular Dystrophy Support Group (N.Z.), Lottery Grants Board of New Zealand, Auckland Medical Research Foundation (N.Z.) and the Health Research Council of New Zealand. References: 1) Monaco.A.P., et al. (1992) Genomics 12, 465-473. 2) Walker.A.P., et al. (1992) Hum. Mol. Genet. 1, 579-585. • To whom correspondence should be addressed
Size (bp) 331 328 325
Frequency 0.07 0.10 0.13
AJlele A4 A5 A6
Size (bp) 322 319 316
Frequency 0.46 0.04 0.20
Calculated heterozygosity: 0.72. Chromosomal Localization: The PK-L-gene has been localized to Iq21 by somatic cell hybrid analysis (2,3). Mendelian Inheritance: Observed in two large two-generation families. Clinical Relevance: Mutations in the human pyruvate kinase L gene cause an autosomal-recessively inherited anemia characterized by reticulocytosis and hemosiderosis. This is the first report of an intragenic polymorphism which can be used for genomic diagnosis of PK-deficiency via linkage analysis. PCR Conditions: PCR amplification was performed in a volume of 25 /tl containing 300 ng genomic DNA, 2.5 /tl Perkin-Elmer 10 x reaction buffer; 200 yM of each dTTP, dATP, dCTP, dGTP; 8.0 pmole of each primer; 2.0 pmole of 5' 32P-labeled primer JHATT; and 1.5 U Taq polymerase. Initial denaturation at 94°C (4 min) was followed by 30 cycles with denaturation at 94°C for 45 sec, annealing at 61°C for 1 min and extension at 72°C for 50 sec. The final extension step was for 7 min. Products were resolved on a 5% sequencing gel and visualized by autoradiography. Allele sizes were estimated by comparison to a M13 sequencing ladder. Acknowledgements: This work was supported by the Deutsche Forschungsgemeinschaft. References: 1) Tani.K. etal. (1988) Proc. Natl. Acad. Sci. USA 85, 1792-1795. 2) Tani.K. etal. (1987)Biochem. Biophys. Res. Commun. 143, 431-438. 3) Satoh.H. etal. (1988) Cytogenet. Cell Genet. 47, 132-133.
* To whom correspondence should be addressed
Downloaded from http://hmg.oxfordjournals.org/ at University of Auckland on December 19, 2011
S.C.King, P.M.Stapleton1, A.P.Walker2 and D.R.Love* School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, 'DNA Diagnostics Ltd, PO Box 5739, Auckland, New Zealand and 2 ICRF Laboratories, Institute of Molecular Medicine, John RadcSffe Hospital, Heacfington, Oxford 0X3 9DU, UK
