Parallel Genotyping of Different Genes:
A Rapid Real-Time PCR Approach
STEFAN FRONHOFFS*, THOMAS BRUNING**, HANS VETTER*, YON Ko*
Introduction
Much progress has been made in identifying clinically relevant point mutations
in genomic DNA. As a result, procedures that allow fast, accurate and easy analysis of known point mutations are needed for diagnosis. To substantially increase
the throughput of sample analysis and/or to analyze individual gene mutation
patterns, it would be helpful to establish the analysis of different parameters in a
parallel procedure.
Hereditary hemochromatosis (HH) is a common autosomal recessive disorder
of iron metabolism, characterized by excessive iron deposition in a variety of
organs leading to multiorgan dysfunction. Two point mutations, G845A and
C187G, referred to as C282Y and H63D, have been detected in the recently identified hemochromatosis gene (HFE) [1]. Most patients (>88%) with HH were found
to be homozygous for the C282Y mutation and a small number are heterozygous
for both the C282Y and the H63D mutations [1,2]. An
point mutation on exon V, resulting in an amino acid change at position 342 [4].
Factor V Leiden, a point mutation (GI691A) in thefactor V gene, has been demonstrated to be one of the most frequent, inherited prothrombotic risk factors [5].
Familial defective apolipoprotein (apo) BlOO is a genetic disorder in which a
reduced affinity of low-density lipoprotein (LDL) to the LDL receptor results in
hypercholesterolemia and premature atherosclerosis [6]. The disorder is caused
by a G~A mutation in the codon for amino acid 3500 [7,8]. Apolipoprotein E
gene (APOE) polymorphism is characterized by three major allelic variants
APOE2, APOE3 and APOE4. APOE3 is considered to be the wild-type allele.
APOE2 is characterized by the point mutation C4070T, which causes an amino
* Stefan Fronhoffs, Hans Vetter, Yon Ko
(~) (e-mail: yonko@uni-bonn.de)
Medizinische Universitats-Poliklinik, WilhelmstraBe 35-37, 53111 Bonn, Germany
** Thomas Briining
Berufsgenossenschaftliches Forschungsinstitut fiir Arbeitsmedizin
an der Ruhr Universitat Bochum, Biirkle-de-la-Camp Platz 1,44789 Bochum, Germany
•
A Rapid Real-Time PCR Approach
STEFAN FRONHOFFS*, THOMAS BRUNING**, HANS VETTER*, YON Ko*
Introduction
Much progress has been made in identifying clinically relevant point mutations
in genomic DNA. As a result, procedures that allow fast, accurate and easy analysis of known point mutations are needed for diagnosis. To substantially increase
the throughput of sample analysis and/or to analyze individual gene mutation
patterns, it would be helpful to establish the analysis of different parameters in a
parallel procedure.
Hereditary hemochromatosis (HH) is a common autosomal recessive disorder
of iron metabolism, characterized by excessive iron deposition in a variety of
organs leading to multiorgan dysfunction. Two point mutations, G845A and
C187G, referred to as C282Y and H63D, have been detected in the recently identified hemochromatosis gene (HFE) [1]. Most patients (>88%) with HH were found
to be homozygous for the C282Y mutation and a small number are heterozygous
for both the C282Y and the H63D mutations [1,2]. An
point mutation on exon V, resulting in an amino acid change at position 342 [4].
Factor V Leiden, a point mutation (GI691A) in thefactor V gene, has been demonstrated to be one of the most frequent, inherited prothrombotic risk factors [5].
Familial defective apolipoprotein (apo) BlOO is a genetic disorder in which a
reduced affinity of low-density lipoprotein (LDL) to the LDL receptor results in
hypercholesterolemia and premature atherosclerosis [6]. The disorder is caused
by a G~A mutation in the codon for amino acid 3500 [7,8]. Apolipoprotein E
gene (APOE) polymorphism is characterized by three major allelic variants
APOE2, APOE3 and APOE4. APOE3 is considered to be the wild-type allele.
APOE2 is characterized by the point mutation C4070T, which causes an amino
* Stefan Fronhoffs, Hans Vetter, Yon Ko
(~) (e-mail: yonko@uni-bonn.de)
Medizinische Universitats-Poliklinik, WilhelmstraBe 35-37, 53111 Bonn, Germany
** Thomas Briining
Berufsgenossenschaftliches Forschungsinstitut fiir Arbeitsmedizin
an der Ruhr Universitat Bochum, Biirkle-de-la-Camp Platz 1,44789 Bochum, Germany
•