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Title: Galactosemia: A strategy to identify new biochemical phenotypes and molecular genotypes

Abstract

We describe a stratagem for identifying new mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. GALT enzyme activity and isoforms were defined in erythrocytes from probands and their first-degree relatives. If the biochemical phenotypes segregated in an autosomal recesssive pattern, we screened for common mutations by using multiplex PCR and restriction endonuclease digestions. If common mutant alleles were not present, the 11 exons of the GALT gene were amplified by PCR, and variations from the normal nucleotide sequences were identified by SSCP. The suspected region(s) was then analyzed by direct DNA sequencing. We identified 86 mutant GALT alleles that reduced erythrocyte GALT activity. Seventy-five of these GALT genomes had abnormal SSCP patterns, of which 41 were sequenced, yielding 12 new and 21 previously reported, rare mutations. Among the novel group of 12 new mutations, an unusual biochemical phenotype was found in a family whose newborn proband has classical galactosemia. He had inherited two mutations in cis (N314D-E204K) from his father, whose GALT activity was near normal, and an additional GALT mutation in the splice-acceptor site of intron C (IVSC) from his mother. The substitution of a positively charged E204K mutation created a unique isoform-banding pattern. An asymptomatic sister`s GALT genesmore » carries three mutations (E203K-N314D/N314D) with eight distinct isoform bands. Surprisingly, her erythrocytes have normal GALT activity. We conclude that the synergism of pedigree, biochemical, SSCP, and direct GALT gene analyses is an efficient protocol for identifying new mutations and speculate that E203K and N314D codon changes produce intra-allelic complementation when in cis. 40 refs., 4 figs., 3 tabs.« less

Authors:
; ; ; ; ; ; ; ; ;  [1]
  1. Emory Univ. School of Medicine, Atlanta, GA (United States)
Publication Date:
OSTI Identifier:
70440
Resource Type:
Journal Article
Resource Relation:
Journal Name: American Journal of Human Genetics; Journal Volume: 56; Journal Issue: 3; Other Information: PBD: Mar 1995
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; GENES; GENE MUTATIONS; DNA SEQUENCING; PATIENTS; HEREDITARY DISEASES; ENZYMES; BIOCHEMISTRY; GENOME MUTATIONS; DIAGNOSIS; GALACTOSE; METABOLISM; POLYMERASE CHAIN REACTION; PHENOTYPE; GENOTYPE

Citation Formats

Elsas, L.J., Langley, S., Steele, E., Evinger, J., Brown, A., Singh, R., Fernhoff, P., Hjelm, L.N., Dembure, P.P., and Fridovich-Keil, J.L. Galactosemia: A strategy to identify new biochemical phenotypes and molecular genotypes. United States: N. p., 1995. Web.
Elsas, L.J., Langley, S., Steele, E., Evinger, J., Brown, A., Singh, R., Fernhoff, P., Hjelm, L.N., Dembure, P.P., & Fridovich-Keil, J.L. Galactosemia: A strategy to identify new biochemical phenotypes and molecular genotypes. United States.
Elsas, L.J., Langley, S., Steele, E., Evinger, J., Brown, A., Singh, R., Fernhoff, P., Hjelm, L.N., Dembure, P.P., and Fridovich-Keil, J.L. Wed . "Galactosemia: A strategy to identify new biochemical phenotypes and molecular genotypes". United States.
@article{osti_70440,
title = {Galactosemia: A strategy to identify new biochemical phenotypes and molecular genotypes},
author = {Elsas, L.J. and Langley, S. and Steele, E. and Evinger, J. and Brown, A. and Singh, R. and Fernhoff, P. and Hjelm, L.N. and Dembure, P.P. and Fridovich-Keil, J.L.},
abstractNote = {We describe a stratagem for identifying new mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. GALT enzyme activity and isoforms were defined in erythrocytes from probands and their first-degree relatives. If the biochemical phenotypes segregated in an autosomal recesssive pattern, we screened for common mutations by using multiplex PCR and restriction endonuclease digestions. If common mutant alleles were not present, the 11 exons of the GALT gene were amplified by PCR, and variations from the normal nucleotide sequences were identified by SSCP. The suspected region(s) was then analyzed by direct DNA sequencing. We identified 86 mutant GALT alleles that reduced erythrocyte GALT activity. Seventy-five of these GALT genomes had abnormal SSCP patterns, of which 41 were sequenced, yielding 12 new and 21 previously reported, rare mutations. Among the novel group of 12 new mutations, an unusual biochemical phenotype was found in a family whose newborn proband has classical galactosemia. He had inherited two mutations in cis (N314D-E204K) from his father, whose GALT activity was near normal, and an additional GALT mutation in the splice-acceptor site of intron C (IVSC) from his mother. The substitution of a positively charged E204K mutation created a unique isoform-banding pattern. An asymptomatic sister`s GALT genes carries three mutations (E203K-N314D/N314D) with eight distinct isoform bands. Surprisingly, her erythrocytes have normal GALT activity. We conclude that the synergism of pedigree, biochemical, SSCP, and direct GALT gene analyses is an efficient protocol for identifying new mutations and speculate that E203K and N314D codon changes produce intra-allelic complementation when in cis. 40 refs., 4 figs., 3 tabs.},
doi = {},
journal = {American Journal of Human Genetics},
number = 3,
volume = 56,
place = {United States},
year = {Wed Mar 01 00:00:00 EST 1995},
month = {Wed Mar 01 00:00:00 EST 1995}
}