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Clinical Chemistry 45, No. 9, 1999
tal intensive care unit is facilitated by the use of
the Ames Glucometer Elite electrochemical glucose meter. J Pediatr 1997;130:151–5.
2. Trajanoski Z, Brunner GA, Gfrerer RJ, Wach P,
Pieber TR. Accuracy of home blood glucose
meters during hypoglycemia. Diabetes Care
1996;19:1412–5.
Laurence M. Demers*
Betty Smith
Section of Clinical Chemistry
The PennState-Geisinger
Health System
The M.S. Hershey Medical Center
Hershey, PA 17033
*Author for correspondence.
Fig. 1. Scattergram comparing blood glucose analysis of 74 consecutive blood samples from our
neonatal unit by the Precision-G (y-axis) vs the Vitros 950 (x-axis).
homeostasis in the newborn is extremely important to good patient
care in the neonatal intensive care
setting, and the laboratory is frequently challenged to provide a
more rapid and sensitive means of
determining blood glucose in the hypoglycemic infant (1 ). Recently, several point-of-care testing instruments
have been developed that allow for
on-site glucose testing at the low end
of the dynamic range of glucose measurements (2 ). We recently evaluated
the Precision-G System by MediSense, which can determine glucose
on a 5-mL blood specimen in 20 s at
the bedside with a dynamic range
that extends to 1.11 mmol/L (20 mg/
dL). Seventy-four consecutive blood
samples from the neonatal unit were
collected by heelstick into lithium
heparin capillary tubes, and glucose
was determined simultaneously on
an Ortho Clinical Diagnostics Vitros
950 analyzer and the Precision-G
System. The neonatal population we
studied demonstrated the usual
range of normal to high hematocrit
values as well as increased bilirubin,
providing assurance that these variables did not influence the results.
Comparative results between these
two methods are shown in Fig. 1.
Regression analysis of the glucose
values obtained with the Precision-G
and the Vitros 950 (Fig. 1) revealed a
correlation coefficient of 0.993 (Syux 5
0.43 mmol/L), an intercept 5 20.053
(6 0.002) mmol/L, and a slope of
0.946 (6 0.051). Although the number of samples in the true hypoglycemic range was rather limited, the
results from 14 subjects with glucose
results below 2.22 mmol/L (40 mg/
dL) fell on the regression line. Overall, there was a slight negative systematic difference observed with the
Precision-G results that averaged
0.31 mmol/L; differences in the analytical methods and the use of whole
blood vs plasma sample most likely
account for this difference. This
slight difference, however, should
have little influence on the neonatologist’s clinical decision regarding
the detection of hypoglycemia. Between-run imprecision (CV) with
the Precision-G using Abbott MediSense control material at 1.11 mmol/L
(20 mg/dL) was 8% (n 5 15). It is our
impression from these results that the
Precision-G System can be used effectively to determine low blood glucose
concentrations in a neonatal unit.
References
1. Innanen VT, Deland ME, de Campos FM, Dunn
MS. Point-of-care glucose testing in the neona-
Uncovering Rare Mutations: An
Unforeseen Complication of Routine
Genotyping of APOE
To the Editor:
APOE genotyping to identify subjects with the E4 allele is helpful in
the diagnosis of Alzheimer disease
when used together with clinical criteria (1 ). The most common APOE
genotyping method involves digestion of a 244-bp PCR-amplified fragment of APOE exon 4 followed by
digestion with endonuclease HhaI
(2 ). The digestion creates a characteristic pattern of DNA bands in electrophoresis gels for each of the three
common APOE alleles (E4, E3, and
E2) and thus for the six common
APOE genotypes (E4/4, E3/3, E2/2,
E4/3, E3/2, and E4/2) (2 ). However,
there are four additional HhaI recognition sites within the 244-bp fragment that is amplified by this
method, and the fragment also harbors several sites that differ from the
HhaI recognition sequence (GCG/C)
by a single nucleotide (2 ).
In the course of .2000 APOE
genotyping reactions, we have observed two individuals who had patterns of HhaI restriction fragments
that were distinct from any that
could have resulted from the common APOE genotypes (Fig. 1). DNA
sequencing of these two individuals
revealed that each was heterozygous
for a different rare APOE mutation,
1580
Letters
Robarts Research Institute
406-100 Perth Dr.
London, Ontario, Canada N6A 5K8
Fax 519-663-3789
E-mail [email protected]
J. Merz and L. Silverman provide the
following comment:
Fig. 1. HhaI restriction endonuclease patterns after digestion of APOE exon 4.
MV is a molecular weight marker, and fragment sizes (bp) are indicated on the right. Lanes 1 and 2 show the
typical fragment pattern resulting from the E3/2 genotype. Lanes 3–5 show the typical fragment patterns
resulting from the E4/3, E3/3, and E4/2 genotypes, respectively. Lanes 6 and 7 each show an atypical HhaI
fragment of 109 bp. DNA sequencing showed that in both cases the 109-bp fragment resulted from the loss
of a HhaI recognition site in codon 136. For the subject in lane 6, a C3 A substitution at the first nucleotide
position of codon 136 produced an Arg (CGC) to Ser (AGC) substitution in one allele. The typical E2 sequence
(C112; C158) was found in the second allele. For the subject in lane 7, a single C3 T substitution at the first
nucleotide position of codon 136 produced an Arg (CGC) to Cys (TGC) substitution in one allele. The typical
E3 sequence (C112; R158) was found in the second allele.
namely R136C and R136S. Both of
these mutations have been reported
previously in probands with hyperlipidemia and premature atherosclerosis (3 ), as was the case for both
subjects in the present report.
The frequency of rare mutations in
APOE has been estimated to be as
high as 1% (3 ). The DNA sequence
change produced by some of these
mutations will create an unusual pattern of HhaI fragments. Thus, the
increased use of APOE genotyping
for Alzheimer disease will likely
identify numerous individuals with
rare mutations in APOE. This means
that the laboratories that perform
APOE genotyping with HhaI will
need to be aware of the possibility of
uncovering rare APOE mutations
and will need to recognize when this
occurs. Furthermore, the APOE genotype would be of questionable
benefit for the diagnosis of Alzheimer disease in a patient with a rare
mutation of APOE. In addition, before ordering the test, the attending
physician would need to be aware of,
and perhaps make the patient and
family aware of, the possibility that
the routine genotyping test might
uncover a new APOE mutation. Finally, the physician who orders the
test would have to be able to interpret the possible pathophysiological
and genetic implications of a rare
APOE mutation when it is found.
These potential problems could be
circumvented by the use of an APOE
genotyping method that is limited
only to the detection of the C3 G
change underlying the R/C112 amino
acid polymorphism underlying the E4
allele (4), thus eliminating the possibility of detecting any other APOE sequence changes.
This work was supported by the Medical Research Council of Canada
(Grant MA13430), the Heart and
Stroke Foundation of Ontario (Grant
3628), and general support from the
Blackburn Group. Dr. Hegele is a Career Investigator of the Heart and
Stroke Foundation of Ontario (CI-2979).
References
1. Mayeux R, Saunders AM, Shea A, Mirra S,
Evans D, Roses AD, et al. Utility of the apolipoprotein E genotype in the diagnosis of Alzheimer’s disease. N Engl J Med 1998;338:506 –
11.
2. Hixson JE, Vernier DT. Restriction isotyping of
human apolipoprotein E by gene amplification
and cleavage with HhaI. J Lipid Res 1991;31:
545– 8.
3. Walden CC, Hegele RA. Apolipoprotein E in
hyperlipidemia. Ann Intern Med 1994; 120:
1026 –36.
4. Emi M, Wu LL, Robertson MA, Myers RL, Hegele
RA, Williams RR, et al. Genotyping and sequence analysis of apolipoprotein E isoforms.
Genomics 1988;3:373–9.
Robert A. Hegele
Blackburn Cardiovascular
Genetics Laboratory
To the Editor:
Dr. Hegele’s finding of unique disease-causing mutations in the APOE
gene during routine genotyping for
Alzheimer disease highlights the
continuing need for broad patientoriented clinical observation and intervention as genetic discoveries and
tests move from the research laboratory into clinical use. This type of
discovery, made in the course of clinical testing, can be stifled by the
monopolization of testing services
enabled by the exclusive licensing of
genetic testing patents (1 ). Indeed,
Dr. Hegele admits in his letter to
performing more than 2000 tests, and
his performance of these tests may
well infringe a Canadian patent
sought by Duke University on
APOE4 testing. Laboratorians in the
US are prevented from making similar discoveries because of the exclusive license on the test granted by
Duke to Athena Diagnostics. The fear
of being sued might also lead clinical
laboratorians not to publish such
findings, which is a foreseeable and
unfortunate result of patents on
medical processes such as tests, and
which would be antithetical to medical and scientific norms.
Perhaps more importantly, Dr. Hegele notes the importance of working
with informed patients (and perhaps
involved family members) to determine the proper scope of testing to
be performed. This example of the
uncertainty and continuing research
nature of clinical discovery about the
role of genetic mutations in Alzheimer disease also supports the assertion that a patent-based monopoly
on clinical testing services unreasonably interferes with both patient care
and science. This will likely become
more of a problem as tests move into
clinical use for the rapidly growing
list of known disease genes.