GENOMICS 13, 7-15 (19%) A Minisatellite and a Microsatellite Polymorphism within 1.5 kb at the Human Muscle Glycogen Phosphorylase (PYGM) Locus Can Be Amplified by PCR and Have Combined Informativeness of PIC 0.95 HIROYUKI IWASAKI, PETER W. STEWART, WILLIAM G. DILLEY, MATTHEW 5. HOLT,* TODD D. STEINBRUECK,* SAMUEL A. WELLS, JR., AND HELEN DONIS-KELLER* Department of Surgery and *Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63170 Received November 18. 1991 We sequenced a genomic clone (pMCMPl), previously re- ported to detect a VNTR polymorphism at the PYGM locus, and found a dinucleotide repeat segment (CA),,(GA),, and a complex (AT)-repeat-rich segment containing 63 repeats spanning 160 bp. Resolution of PCR-amplified genomic DNA from the (CA)(GA) repeat region on DNA sequencing gels re- vealed a highly informative polymorphism with alleles differ- ing by Z-bp intervals and ranging in size from 156 to 190 bp. Among three racial groups, a total of 18 alleles were observed. Fourteen alleles were observed in Caucasians (PIC O&9), 12 alleles in American Blacks (PIC 0.89), and 9 alleles in Pima Indians (PIC 0.73). PCR amplification of the (AT) repeat re- gion and resolution of the products on DNA sequencing gels revealed a complex variable length polymorphism with alleles distributed in size from 367 to 970 bp. Twenty-eight alleles were found in American Blacks (PIC 0.94), 6 alleles in Pima Indians (PIC 0.70), and 11 alleles in Caucasians (PIC 0.71). Comparison of the previously described VNTR RFLP alleles visualized by Southern hybridization to the PCR products de- scribed in this report demonstrated that the polymorphism de- tected in both assays was identical. However, a larger number of alleles could be detected from the PCR-amplified products. Combined informativeness, PIC 0.95, for the two polymor- phisms was determined from haplotype analysis of 100 Cau- casian chromosomes. Therefore, for genotyping purposes, in- formativeness is maximized from using both polymorphisms. The sequence of the PYGM 2.1-kb cloned insert has been de- posited with the GenBank Data Libraries (Accession No. M77201). 0 1992 Academic Press, Inc. INTRODUCTION Once genetic linkage is found between a disease locus and a genetic marker, fine structure genetic mapping studies, including the identification of affected individ- uals recombinant for the nearest flanking markers, pro- vide crucial information for designing gene cloning strat- egies and for development of predictive DNA diagnostic tests. Poor informativeness of genetic markers and diffi- culties in making accurate allele assignments severely limit the usefulness of fine structure genetic mapping approaches for these purposes. For example, the gene for human muscle glycogen phosphorylase (PYGM) has been reportedly linked, with no observed recombinants, to the autosomal and dominantly inherited disease syn- drome, multiple endocrine neoplasia type I (MEN-l) (Larsson et aZ., 1988; Nakamura et aZ., 1989; Bystrom et al., 1990). The PYGM gene has been mapped to llq13 (Lebo et al., 1984; Glaser et aZ., 1988), and a VNTR poly- morphism has been characterized from a genomic cos- mid clone identified by a PYGM cDNA clone (Carlson et al., 1988). However, the RFLP marker offers relatively low informativeness (56% heterozygosity, PIC 0.46) and presents difficulty in genotyping because the reported 3 MspI alleles are less than 2.5 kb in length and differ in size by about 100 bp each, which makes them extremely difficult to resolve by conventional agarose gel electro- phoresis and Southern transfer hybridization. Seeking a more informative marker in the region of the PYGM gene, we tested the cloned VNTR probe for the presence of (CA) repeats of genomic DNA. Such di- nucleotide repeat elements have been reported to be rela- tively abundant in the genome, averaging one CA repeat per 30-60 kb of genomic DNA (Hamada and Kakunaga, 1982). Many of these elements are highly informative genetic markers and offer the additional advantage of assay by PCR methods (Weber and May, 1989; Litt and Luty, 1989; Weber, 1990). We report the identification of two polymorphic dinucleotide repeat elements (one of which contains a VNTR element) that substantially in- crease the informativeness of the PYGM locus. MATERIALS AND METHODS Subjects. Human genomic DNA from 40 CEPH reference pedi- grees was obtained from the Centre d’Etude du Polymorphisme Hu- main, Paris, or was prepared from cell lines in-house. DNAs from 25 unrelated American Pima Indians (Clifton Bogardus, NIH, Phoenix, AZ) and 25 unrelated American Blacks were graciously provided by Dr. M. Alan Permutt, Department of Internal Medicine, Washington University School of Medicine. Signed consents were obtained ac- cording to a protocol approved by the Human Studies Committee at Washington University. 7 08&3-7543/z $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.