Analysis of the preproPTH gene by denaturing gradient gel electrophoresis in familial isolated hypoparathyroidism

Alexander Miric, Michael A. Levine

    Research output: Contribution to journalArticle

    Abstract

    Familial isolated hypoparathyroidism (FIH) is an inherited metabolic disorder characterized by hypocalcemia and hyperphosphatemia due to deficient secretion of biologically active PTH. We used denaturing gradient gel electrophoresis to screen for mutations in exon 1 and the coding region of the preproPTH gene. Exons 1, 2, and 3 of the preproPTH gene and flanking intronic regions were amplified by polymerase chain reaction using primers that were designed employing the MELT-MAP program. One oligonucleotide from each primer pair was synthesized with a 5′-GC-clamp. Screening of amplified DNA from normal subjects and patients with FIH revealed two single base changes that altered migration of amplified preproPTH gene fragments through denaturing gels: 1) an A → G transition in intron 1; 10 nucleotides upstream of exon 2; and 2) a C → A transversion in exon 3 that conserves the arginine residue at codon 52 (CGA → AGA). By contrast, we did not detect pathogenic mutations in amplified regions of the preproPTH genes of 18 affected members of 5 FIH kindreds. The two polymorphisms occur frequently, and were therefore used to perform linkage analysis in 5 multiplex FIH families. Linkage analysis was inconclusive in 2 families and showed discordance between hypoparathyroidism and any of the preproPTH gene alleles in 2 other families. In another family, analysis was suggestive of linkage between hypoparathyroidism and inheritance of a specific preproPTH gene allele. These results indicate that denaturing gradient gel electrophoresis can be used to identify mutations in defined regions of the preproPTH gene, and to examine linkage of specific preproPTH alleles and inherited disorders of mineral metabolism.

    Original languageEnglish (US)
    Pages (from-to)509-516
    Number of pages8
    JournalJournal of Clinical Endocrinology and Metabolism
    Volume74
    Issue number3
    StatePublished - Mar 1992

    Fingerprint

    Denaturing Gradient Gel Electrophoresis
    Electrophoresis
    Genes
    Gels
    Exons
    Hypoparathyroidism
    Alleles
    Mutation
    Hyperphosphatemia
    DNA Primers
    Hypocalcemia
    Polymerase chain reaction
    Clamping devices
    Hypoparathyroidism familial isolated
    Polymorphism
    Metabolism
    Codon
    Introns
    Minerals
    Arginine

    ASJC Scopus subject areas

    • Biochemistry
    • Endocrinology, Diabetes and Metabolism

    Cite this

    Analysis of the preproPTH gene by denaturing gradient gel electrophoresis in familial isolated hypoparathyroidism. / Miric, Alexander; Levine, Michael A.

    In: Journal of Clinical Endocrinology and Metabolism, Vol. 74, No. 3, 03.1992, p. 509-516.

    Research output: Contribution to journalArticle

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    abstract = "Familial isolated hypoparathyroidism (FIH) is an inherited metabolic disorder characterized by hypocalcemia and hyperphosphatemia due to deficient secretion of biologically active PTH. We used denaturing gradient gel electrophoresis to screen for mutations in exon 1 and the coding region of the preproPTH gene. Exons 1, 2, and 3 of the preproPTH gene and flanking intronic regions were amplified by polymerase chain reaction using primers that were designed employing the MELT-MAP program. One oligonucleotide from each primer pair was synthesized with a 5′-GC-clamp. Screening of amplified DNA from normal subjects and patients with FIH revealed two single base changes that altered migration of amplified preproPTH gene fragments through denaturing gels: 1) an A → G transition in intron 1; 10 nucleotides upstream of exon 2; and 2) a C → A transversion in exon 3 that conserves the arginine residue at codon 52 (CGA → AGA). By contrast, we did not detect pathogenic mutations in amplified regions of the preproPTH genes of 18 affected members of 5 FIH kindreds. The two polymorphisms occur frequently, and were therefore used to perform linkage analysis in 5 multiplex FIH families. Linkage analysis was inconclusive in 2 families and showed discordance between hypoparathyroidism and any of the preproPTH gene alleles in 2 other families. In another family, analysis was suggestive of linkage between hypoparathyroidism and inheritance of a specific preproPTH gene allele. These results indicate that denaturing gradient gel electrophoresis can be used to identify mutations in defined regions of the preproPTH gene, and to examine linkage of specific preproPTH alleles and inherited disorders of mineral metabolism.",
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