Biomedical Engineering Reference
In-Depth Information
dhplc_genes2.html).
In this chapter, we use the
G6PT1
gene as an example to
illustrate the use of DHPLC for prenatal diagnosis of a genetic disease
(
7
)
(
see
Note 1
).
Glycogen storage disease Ib (GSD Ib) is an inborn error of metabolism with
autosomal recessive inheritance, caused by a deficiency in glucose-6-phosphate
translocase (G6PT1). Patients with GSD Ib present with hypoglycemia,
hepatomegaly, kidney enlargement, growth retardation, lactic acidemia, hyper-
lipidemia, hyperuricemia, neutropenia and impaired neutrophil function, oral
and intestinal mucosa ulcerations, and inflammatory bowel disease. The G6PT1
protein has not been purified to homogeneity, and its molecular nature remained
unknown until recently.
Gerin et al.
(
10
)
, using a candidate gene approach, identified mutations of a
putative human
G6PT1
, homologous to bacterial transporters of hexose-6-
phosphate, in patients with GSD Ib. The gene is composed of nine exons span-
ning a genomic region of approx 5 kb
(
11
)
. The liver cDNA encodes a protein
of 429 amino acids. We have identified a patient clinically suspected of GSD
Ib
(
12
)
. The patient has all of the clinical features of GSD Ib, including neutro-
penia. The patient is the only child in the family, and the parents are not con-
sanguineous. DNA sequencing of all nine exons and the exon-intron boundaries
revealed a homozygous G>A transition at nucleotide position 1563 of the
genomic DNA sequence. The missense mutation is located in exon 3, at codon
149, changing the nonpolar glycine to the charged glutamic acid, i.e., G149E.
The father and mother are each heterozygous for this mutation. In this study,
we have used DHPLC for prenatal diagnosis of this disease in her second
pregnancy.
2. Materials
1.
Genomic DNA template.
2.
QIAamp Tissue Kit (Qiagen).
3.
PCR primers.
4.
PCR kit (
see
Note 2
).
5.
Thermal block or water bath.
6.
WAVE™ DHPLC analyzer and software (Transgenomic Inc., San Jose, CA).
7.
DNASep cartridge packed with C18 alkylated, polystyrene-divinylbenzene poly-
meric beads.
8.
Eluent A: 0.1
M
triethylammonium acetate (TEAA).
9.
Eluent B: 0.1
M
TEAA and 25% acetonitrile (ACN).
3. Methods
3.1. PCR Amplification
1.
Genomic DNA is extracted from cultured chorionic villi using a QIAamp Tissue
Kit (Qiagen) according to the manufacturer's instructions.
